Compositions and methods for immune cell modulation in adoptive immunotherapies

ABSTRACT

Compounds that either produced a higher proportion or greater absolute number of phenotypically identified nave, stem cell memory, central memory T cells, adaptive NK cells, and type I NKT cells are identified. Compositions and methods for modulating immune cells including T, NK, and NKT cells for adoptive cell therapies with improved efficacy are provided.

RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 62/281,064, filed Jan. 20, 2016 and U.S. Provisional PatentApplication No. 62/402,883, filed Sep. 30, 2016, the disclosures ofwhich are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present disclosure is broadly concerned with the field of adoptiveimmune cell therapies. More particularly, the present disclosure isconcerned with the use of small molecules for modulating immune-cellssuitable for adoptive cell therapies.

BACKGROUND OF THE INVENTION

Adoptive immunotherapy involves administration of immune cells topatients having cancer, tumor, or infections, whereby the administeredimmune cells provide a therapeutic benefit to the patients. Generallyspeaking, immune cells suitable for immunotherapy include, but are notlimited to, B cells, dendritic cells (DC), T cells, Natural Killer (NK)cells, NKT (Natural Killer T) cells, and hematopoietic stem orprogenitor cells. Mediating complete and durable disease responses inpatients is the central goal of these cell-based immunotherapies.

Advances in our understanding of the biological mechanisms behind theeffectiveness of adoptive T cell therapies, including, but not limitedto, CAR-T cells, TCR-T cells, virus-specific T cells (VSTs) andtumor-infiltrating T cell (TILs), have underscored the importance of thecertain attributes associated with transferred T cells and revealed thecomplexity of the inhibitory barriers posed by the host and tumor cellsthat need to be overcome for the success of the treatment of cancer.Among T cell factors, the avidity of the T cell receptor (TCR) orchimeric antigen receptor (CAR), the proliferative and survivalcapacities, migration to the tumor site(s), and the ability to sustaineffector functions within the tumor have, in correlative studies, beenshown to be crucial determinants for triggering the eradication ofmalignant cells. However, adding to another layer of complexity, eventhough some of these desirable attributes were recognized, the pathwaysor players driving these attributes are still unclear, which limitsone's ability to intervene and obtain cells having desired quantity andquality for their therapeutic uses.

Using CAR-T cell therapy as an example, the therapy has to overcomemultiple challenges including CAR-T potency and persistence, migrationto the tumor, the immunosuppressive tumor microenvironment, tumorheterogeneity and patient safety. Multiple approaches are being appliedto overcome these challenges. For example, specific T-cell subsets areselected for therapeutic use and further engineering of the CAR may beused to improve tumor targeting, CAR potency and on-target/off-tumorsafety issues. However, improving the CAR-T therapeutic efficacy,including CAR-T persistence and migration remains to be resolved. It hasbeen shown that the in vivo efficacy of the T cell therapy can bestrongly influenced by the manufacturing process which is dependent uponboth the starting population of T cells going into the process orfeedstock, and the ex vivo expansion and activation methods utilized. Ithas been demonstrated that the differentiation state of the administeredT cells can significantly affect in vivo persistence and anti-tumoractivity. T helper (CD4+ T cells) and cytotoxic T cells (CD8+),specifically, naïve (Tn), stem cell memory (Tscm) and central memory(Tcm) T cells, characterized by the expression of the CCR7 and CD62Lmarkers, mediate superior anti-tumor activity in both mouse models(Sommermeyer et al. 2015) and in nonhuman primate models (Berger et al.2008).

During the manufacturing process, therapeutic cells (or cellpopulations) are typically activated and expanded. This processgenerally drives differentiation of the cells and leads to an increasein the proportion of the cells in a more differentiated state—in thecase of T-cells, the more differentiated cells are phenotypicallycharacterized as effector memory or effector T cells. Once infused intopatients, these more differentiated cells have a lower capacity toproliferate and a lower potential to persist as a long-lived orpersistent population, as compared to cells in less differentiatedstates. Thus, there is an urgent need in the art not only forcompositions and methods useful for maintaining and expanding desiredimmune cell subsets, but also for reducing cell differentiation duringexpansion, and for dedifferentiating cells to less differentiated cells,thereby obtaining desired immune cell subsets that have greater capacityto proliferate and persist in order to improve the efficacy of variousadoptive immunotherapies.

Similar efficacy issues exist in NK-cell based therapies as well.Natural killer cells have traditionally been categorized as innateimmune cells that are characterized as being relatively short-lived andexhibit minimal change in response to secondary exposure to a stimulusi.e., display limited target memory responses. However, recent researchhas uncovered information on both activating and inhibitory NK cellreceptors which play important roles including self-tolerance andsustaining NK cell activity. Data have demonstrated the ability of NKcells to readily adjust to the immediate environment and formulateantigen-specific immunological memory, which is fundamental forresponding to secondary exposure to the same antigen. A subpopulation ofNK cells, now called adaptive NK cells or memory NK cells, have beenidentified by several groups. These cells have many functionalcharacteristics similar to CD8+ T cells, including being longer-livedand having enhanced response to stimuli after an initial exposure. Theseproperties may result in a more efficacious cell therapy strategy, ascompared to canonical NK cells. Expanding and maintainingadaptive/memory NK cells that mediate durable antigen-specificrecognition in vivo would be a key to improving NK-cell based adoptiveimmunotherapy.

Further, it is believed that, like T and NK cells, improvements can bemade to isolate more efficacious NKT cells, a type of CDld-restricted Tcell playing a role in both the innate and adaptive immune systems,which can be targeted for modulation to yield an improved cell therapy.

Since the final state of the cells, or specifically, the cell subtypes,going into the patient can be defined in large part by the manufacturingprocess, the importance of that process cannot be overstated.Preferentially maintaining or expanding cell subpopulations having adesired differentiation state, and/or adaptive immune cellcharacteristics during cell culture and expansion could be extremelybeneficial for enhancing the efficacy of cell-based therapies. Thus, amanufacturing approach that can enhance the desired T, NK or NKT cellsubsets both in quantity and quality could provide a significantenhancement of their therapeutic efficacy.

There is a substantial need in the art for immune cell subsets withimproved therapeutic efficacy. However, while certain desirableattributes of therapeutic immune cells are known, the pathways and/orplayers involved in achieving these attributes are largely unknown. Themethods and compositions of the present invention addresses this needand provide other related advantages in the field of immune celltherapy.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for modulatingone or more populations or subpopulations of immune cells to improvetheir therapeutic potential for adoptive immunotherapies. It is anobject of the present invention to provide one or more compounds, eitheralone or in combination to improve proliferation, persistence,cytotoxicity, and/or cell recall/memory of therapeutic immune cells by,for example, increasing the number or ratio of a subpopulation of cellsthat displays improvement in at least one of the following qualitiesthat are expected to result in better immunotherapeutic results:migration, homing, cytotoxicity, maintenance, expansion, persistence,longevity, desired states of differentiation.

One aspect of the invention provides a composition for improvingtherapeutic potential of immune cells suitable for adoptive cell-basedtherapies, and the composition comprises one or more agents selectedfrom the group consisting of compounds listed in Table 1: Dorsomorphin;Heptelidic acid; 1-Pyrrolidinecarbodithioic acid, ammonium salt;2-dexoyglucose (2-DG); GSK3 Inhibitor; Rho kinase inhibitors; MEKinhibitors; PDK1 agonist; TGFβ inhibitors; 6-Mercaptopurine; AC-93253iodide; Tiratricol; PI-103; Fulvestrant; Thapsigargin; SU 4312;Telmisartan; Cyclosporin A;1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole; BAY 61-3606;Protoporphyrin IX disodium; mTOR inhibitor; HS173; LY294002; Pictilisib;5-Azacytidine; Fludarabine; Roscovitine, (S)-Isomer; PAC-1;8-Quinolinol, 5,7-dichloro-; Nitrofurantoin; 8-Quinolinol,5-chloro-7-iodo-; 2-Naphthacenecarboxamide,7-chloro-4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahy; Nifuroxazide;Tosufloxacin hydrochloride; Sertraline; Diethylenetriaminepentaaceticacid, penta sodium; Edrophonium chloride; BIX01294; Terfenadine; anddmPGE2. The one or more agents selected from the group consisting ofcompounds listed in Table 1 improve therapeutic potential of immunecells or one or more subpopulation thereof via modulating of the immunecells using one or more agent. In some embodiments, the modulation ofthe immune cells is ex vivo.

In some embodiments, the one or more of compounds listed in Table 1modulates cell expansion, maintenance and/or differentiation, andthereby improve proliferation, cytotoxicity, cytokine response andsecretion, cell recall, and/or persistence of the immune cells, or oneor more subpopulation thereof.

In one embodiment, the one or more of the compounds listed in Table 1improves cell survival rate of the immune cell, or one or moresubpopulation thereof both ex vivo and in vivo.

In one embodiment, the one or more of the compounds listed in Table 1increases the ratio of one or more desired cell subpopulation of theimmune cells.

In some embodiments, the present invention provides one or more selectedagents herein to improve therapeutic efficacy of a population orsubpopulation of immune cells, including but not limited to T, NK andNKT cells. In some embodiments, the immune cells immune cells suitablefor adoptive cell-based therapies comprise T cells, NKT cells, or NKcells. In some embodiments, the immune cells subject to the treatmentscomprise T cells, as such the one or more desired cell subpopulationshas an increased ratio comprises naïve T cells, stem cell memory Tcells, and/or central memory T cells. In some embodiments, the immunecells subject to the treatments using the agents comprise NKT cells, assuch the one or more desired cell subpopulations has an increased ratiocomprise type I NKT cells. In some other embodiments, the immune cellssubject to the treatments using the agents comprise NK cells, andwherein the one or more desired cell subpopulations has an increasedratio comprise adaptive NK cells.

In some embodiments, the composition comprising one or more agentsselected from the group consisting of the compounds, or derivatives,analogues or pharmaceutically acceptable salts thereof, listed inTable 1. The compounds, or derivatives, analogues or pharmaceuticallyacceptable salts thereof further comprise ester, ether, solvate,hydrate, stereoisomer, and prodrug of the compounds of Table 1.

In some embodiments, the composition for improving therapeutic potentialof immune cells comprises at least one agent selected from Group I, andone or more agents selected from Group II, Group III, Group IV, and/orGroup V.

Group I comprises: dorsomorphin, heptelidic acid,1-Pyrrolidinecarbodithioic acid, and 2-DG. Without being limited to thetheory, Group I agents, among other potential roles, impact cellmetabolism and nutrient sensing.

Group II comprises: GSK3 Inhibitor, ROCK inhibitor, TGFβ receptorinhibitor, MEK inhibitor, PDK1 agonist, 6-Mercaptopurine, AC-93253iodide, tiratricol, PI-103, fulvestrant, thapsigargin, SU 4312, U0126,telmisartan, cyclosporin A,1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, BAY 61-3606,protoporphyrin IX disodium, mTOR inhibitor, TWS119, HS173, LY294002, andPictilisib. Without being limited to the theory, Group II agents, amongother potential roles, impact signal transduction in various functionalpathways.

Group III comprises: 5-Azacytidine, fludarabine, roscovitine, and PAC-1.Without being limited to the theory, Group III agents, among otherpotential roles, impact cell proliferation and apoptosis.

Group IV comprises: 5,7-dichloro-8-Quinolinol, 2-Naphthacenecarboxamide,7-chloro-4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahy, Nifuroxazide,and Tosufloxacin hydrochloride. Without being limited to the theory,Group IV agents, among other potential roles, may impact cell propertiesrelating to infective processes.

Group V comprises: sertraline, diethylenetriaminepentaacetic acid,edrophonium chloride, BIX01294, terfenadine, and dmPGE2. Without beinglimited to the theory, Group V agents, among other potential roles,generally impact other cell properties relating to expansion,maintenance, differentiation, proliferation, survival rate,cytotoxicity, cell recall, and/or persistence.

In some other embodiments, the composition for improving therapeuticpotential of immune cells comprises at least one agent selected fromGroup II, and one or more agents selected from Group I, Group III, GroupIV, and/or Group V.

In still other embodiments, the composition for improving therapeuticpotential of immune cells comprises at least one agent selected fromGroup III, and one or more agents selected from Group I, Group II, GroupIV, and/or Group V.

In yet other embodiments, the composition for improving therapeuticpotential of immune cells comprises at least one agent selected fromGroup IV, and one or more agents selected from Group I, Group II, GroupIII, and/or Group V.

In still some other embodiments, the composition for improvingtherapeutic potential of immune cells comprises at least one agentselected from Group V, and one or more agents selected from Group I,Group II, Group III, and/or Group IV.

In some embodiments, the composition for improving therapeutic potentialof immune cells comprises a combination comprising at least one agentselected from the group consisting of TWS119, HS173, LY294002,Pictilisib, and 2-DG; and one or more additional agent selected from thegroup consisting of compounds listed in Table 1. In some particularembodiments, the composition comprises a synergistic combination of twoor more agents selected from the group consisting of TWS119, HS173,LY294002, Pictilisib, and 2-DG.

In some embodiments, the composition comprising one or more agentsselected from the group consisting the compounds listed in Table 1further comprises at least one organic solvent selected from the groupconsisting of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF),dimethoxyethane (DME), dimethylacetamide, ethanol and combinationsthereof.

In some embodiments, the composition comprises at least one agentselected from Group II. In some embodiments, the composition comprisesan mTOR inhibitor. In some embodiments, the composition comprises atleast one agent selected from Group II, and one or more agents selectedfrom Group V. In one embodiment, the composition comprises an mTORinhibitor, and dmPGE2, or an analogue or a derivative of dmPGE2. In someembodiments, the mTOR inhibitor is selected from rapamycin, andanalogues or derivatives thereof, which comprise sirolimus, sirolimusderivatives, temsirolimus, 40-O-(2-hydroxy)ethyl-rapamycin (everolimus),40-O-(3-hydroxy)propyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, 40-O-tetrazole-rapamycin, orother O-alkylated and O-methylated rapamycin derivatives. In oneembodiment, dmPGE2 is 16,16-dimethyl Prostaglandin E2. In some otherembodiments, the analogue or derivative of dmPGE2 is selected from thegroup consisting of PGE₂, 16,16-dimethyl PGE₂ p-(p-acetamidobenzamido)phenyl ester, 11-deoxy-16,16-dimethyl PGE₂, 9-deoxy-9-methylene-16,16-dimethyl PGE₂, 9-deoxy-9-methylene PGE₂, 9-keto Fluprostenol, 5-transPGE₂, 17-phenyl-omega-trinor PGE₂, PGE₂ serinol amide, PGE₂ methylester, 16-phenyl tetranor PGE₂, 15(S)-15-methyl PGE₂, 15(R)-15-methylPGE₂, 8-iso-15-keto PGE₂, 8-iso PGE2 isopropyl ester,8-iso-16-cyclohexyl-tetranor PGE₂, 20-hydroxy PGE₂, 20-ethyl PGE₂,11-deoxy PGEi, nocloprost, sulprostone, butaprost, 15-keto PGE₂, and 19(R) hydroxy PGE₂. In one particular embodiment, the compositioncomprises rapamycin and dmPGE2.

Another aspect of the invention provides a composition comprising apopulation or subpopulation of immune cells, and one or more agentsselected from the group consisting of the compounds listed in Table 1,and derivatives and analogues thereof. In some embodiments, the immunecells are contacted with the one or more agents to improve therapeuticpotential of the immune cells for adoptive cell therapy in comparison toimmune cells without such contact. In some embodiments, the immune cellsare contacted with the one or more agents to improve cell expansion,maintenance, differentiation, dedifferentiation, and/or survival rate incomparison to immune cells without the same treatment. In yet some otherembodiments, the immune cells are contacted with the one or more agentsto improve cell proliferation, cytotoxicity, persistence, and/or recallin comparison to immune cells without the same treatment.

In some embodiments, the immune cells contacted with the one or moreagents have an increased number or ratio of a desired subpopulation ofthe immune cells in comparison to immune cells without the sametreatment. In some embodiments, the immune cells comprise T, NK or NKTcells. In one embodiment, the composition comprises a population of Tcells, as such the desired subpopulation of immune cells aftercontacting the agent(s) comprise naïve T cells, stem cell memory Tcells, and/or central memory T cells. In some embodiments, thecomposition comprises a population of NKT cells, as such the desiredsubpopulation of immune cells after contacting the agents comprise typeI NKT cells. In yet some other embodiments, the immune cells comprise apopulation of NK cells, as such the desired subpopulation of immunecells after contacting the agents comprise adaptive NK cells. In otherembodiments the adaptive NK cells comprise CD57+ and at least one ofNKG2C+, low PLZF, low SYK, low FεFRγ, low EAT-2, low TIGIT, low PD1, lowCD7, low CD161, high LTLRB1, high CD45RO, and low CD45RA

In some embodiments, the population or subpopulation of immune cells ofthe composition are isolated from or comprised in peripheral blood, bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, or tumors of asubject. The subject may be healthy, may have an autoimmune disease, ahematopoietic malignancy, a virus infection or a solid tumor, or mayhave been previously administered with genetically modified immunecells. In some embodiments, the subject may be CMV seropositive. In someother embodiments, the isolated immune cells for modulation aregenetically modified (genetically engineered or naturally derived fromrearrangements, mutations, genetic imprinting and/or epigeneticmodification). In some embodiments, the isolated immune cells formodulation comprise at least one genetically modified modality. In someembodiments, the isolated population of immune cells are genomicallyengineered and comprise an insertion, a deletion, and/or a nucleic acidreplacement. In some particular embodiments, the immune cells comprisean exogenous nucleic acid encoding a T Cell Receptor (TCR), a ChimericAntigen Receptor (CAR), and/or overexpression of CD16 or a variantthereof. As such, the genetically modified immune cells are isolated forex vivo modulation using the present compositions and methods asdisclosed. In some embodiments, after modulation, the geneticallymodified immune cells isolated from a subject may be administered to thesame donor or a different patient.

In yet another embodiment, the immune cells are differentiated in vitrofrom stem cells, hematopoietic stem or progenitor cells, or progenitorcells; or are trans-differentiated in vitro from non-pluripotent cellsof hematopoietic or non-hematopoietic lineage. In some embodiments, theimmune cells of the composition are genomically engineered and comprisean insertion, a deletion, or a nucleic acid replacement (substitution,or indel). In some particular embodiments, the immune cells of thecomposition comprise an exogenous nucleic acid encoding a T CellReceptor (TCR) and/or a Chimeric Antigen Receptor (CAR). In someembodiments, the immune cells isolated from tissue of a subject aregenetically engineered, and may comprise a TCR or a CAR. In someembodiments, the immune cells isolated from tissue or a subject is aCAR-T cell.

In still some other embodiments, the immune cells of the composition aredifferentiated in vitro from stem cells, hematopoietic stem orprogenitor cells, or progenitor cells. In one embodiment, the stem cellis induced pluripotent stem cells (iPSCs) or embryonic stem cells(ESCs). In one embodiment, the progenitor cell is a CD34+ hemogenicendothelium cell, a multipotent progenitor cell, a T cell progenitorcell, a NK progenitor cell, or a NKT progenitor cell. In someembodiments, the stem cell, hematopoietic stem or progenitor cell, orprogenitor cell is genomically engineered and comprises an insertion, adeletion, or a nucleic acid replacement, or comprises at least onegenetically modified modality. In one particular embodiment, the stemcell, hematopoietic stem or progenitor cell, or progenitor comprises anexogenous nucleic acid encoding a T Cell Receptor (TCR), a ChimericAntigen Receptor (CAR), and/or overexpression of CD16. In some otherembodiments, the immune cells of the composition aretrans-differentiated in vitro from non-pluripotent cells ofhematopoietic or non-hematopoietic lineage. In some embodiments, thedesired subpopulation of immune cells after modulation comprises immunecells having at least one genetically modified modality. In someembodiments, the genetically modified modality comprises at least one ofsafety switch proteins, targeting modalities, receptors, signalingmolecules, transcription factors, pharmaceutically active proteins andpeptides, drug target candidates; or proteins promoting engraftment,trafficking, homing, viability, self-renewal, persistence, immuneresponse regulation and modulation, and/or survival of the immune cells.In some other embodiments, the genetically modified modalities compriseone or more of (i) deletion or reduced expression of B2M, TAP1, TAP2,Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and anygene in the chromosome 6p21 region; and (ii) introduced or increasedexpression of HLA-E, HLA-G, HACD16, hnCD16, 41BBL, CD3, CD4, CD8, CD47,CD113, CD131, CD137, CD80, PDL1, A2AR, Fc receptor, or surfacetriggering receptors for coupling with bi- or multi-specific oruniversal engagers.

In some embodiments, the composition comprising the immune cells and oneor more agents selected from the group consisting of compounds listed inTable 1, further comprises one of more additional additives selectedfrom the group consisting of peptides, cytokines, mitogens, growthfactors, small RNAs, dsRNAs (double stranded RNAs), mononuclear bloodcells, feeder cells, feeder cell components or replacement factors,vectors comprising one or more polynucleic acids of interest; anantibody, or an antibody fragment; and a chemotherapeutic agent, aradioactive moiety, or an immunomodulatory drug (IMiD). In some of theseembodiments, the antibody, or antibody fragment, specifically binds to aviral antigen. In other embodiments, the antibody, or antibody fragment,specifically binds to a tumor antigen. In some embodiments, theadditional additive comprises. Chemotherapeutic agent refers tocytotoxic antineoplastic agents, that is, chemical agents whichpreferentially kill neoplastic cells or disrupt the cell cycle ofrapidly-proliferating cells, or which are found to eradicate stem cancercells, and which are used therapeutically to prevent or reduce thegrowth of neoplastic cells. Chemotherapeutic agents are also sometimesreferred to as antineoplastic or cytotoxic drugs or agents, and are wellknown in the art.

In one specific embodiment, the composition comprises a mixture of apopulation or subpopulation of immune cells and one or more of a GSK3inhibitor, a TGFβ receptor inhibitor, a ROCK inhibitor, a MEK inhibitor,a PDK1 agonist, and an mTOR inhibitor, wherein the immune cells compriseT cells, NK cells or NKT cells. In one embodiment, the compositioncomprises an mTOR inhibitor, wherein the immune cells comprise T cells.In one embodiment, the T cells comprise CAR-T cells. In someembodiments, the mTOR inhibitor is selected from rapamycin, andanalogues or derivatives thereof.

In some embodiments, the composition comprising the immune cells and oneor more agents listed in Table 1 comprises at least one agent selectedfrom Group II, and one or more agents selected from Group V. In oneembodiment, the composition comprises an mTOR inhibitor and dmPGE2, oran analogue or derivative of dmPGE2. In some embodiments, the mTORinhibitor is selected from rapamycin, and analogues or derivativesthereof, which comprise sirolimus, sirolimus derivatives, temsirolimus,40-O-(2-hydroxy)ethyl-rapamycin (everolimus),40-O-(3-hydroxy)propyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, 40-O-tetrazole-rapamycin, andother O-alkylated or O-methylated rapamycin derivatives. In oneembodiment, dmPGE2 is 16,16-dimethyl Prostaglandin E2. In some otherembodiments, the analogue or derivative of dmPGE2 is selected from thegroup consisting of PGE₂, 16,16-dimethyl PGE₂ p-(p-acetamidobenzamido)phenyl ester, 1 l-deoxy-16,16-dimethyl PGE₂, 9-deoxy-9-methylene-16,16-dimethyl PGE₂, 9-deoxy-9-methylene PGE₂, 9-keto Fluprostenol, 5-transPGE₂, 17-phenyl-omega-trinor PGE₂, PGE₂ serinol amide, PGE₂ methylester, 16-phenyl tetranor PGE₂, 15(S)-15-methyl PGE₂, 15(R)-15-methylPGE₂, 8-iso-15-keto PGE₂, 8-iso PGE2 isopropyl ester,8-iso-16-cyclohexyl-tetranor PGE₂, 20-hydroxy PGE₂, 20-ethyl PGE₂,11-deoxy PGEi, nocloprost, sulprostone, butaprost, 15-keto PGE₂, and 19(R) hydroxy PGE₂. In one particular embodiment, the compositioncomprises CAR-T cells, rapamycin and dmPGE2.

Still, another aspect of the invention provides a composition comprisingan isolated population of immune cells that has been contacted ormodulated with a composition comprising one or more agents listed inTable 1, or a derivative or an analogue thereof. In some embodiments,the composition provided is a therapeutic composition having the treatedisolated population or subpopulation of immune cells including, but notlimited to, T, NK, and NKT cells. The therapeutic composition can bewashed with a buffer substantially free of the modulating agent.

In some embodiments, the modulated cell population comprises immunecells having improved therapeutic potential for adoptive cell therapy incomparison to unmodulated cell population. In some embodiments, theisolated population of immune cells has improved cell expansion,maintenance, differentiation, dedifferentiation, and/or survival rate incomparison to immune cells without the treatment by the one or moreagents. In some embodiments, the isolated population of immune cells hasimproved cell proliferation, cytotoxicity, cytokine response andsecretion, cell recall, and persistence in comparison to immune cellswithout the treatment by the one or more agents. In some otherembodiments, the isolated population of immune cells has increasednumber or ratio of one or more desired subpopulations of the immunecells in comparison to immune cells without the same treatment.

In some embodiments, the isolated population of immune cells thattreated with one or more agents selected from the group consisting ofcompounds listed in Table 1 comprises T cells, as such the obtained oneor more desired subpopulation of immune cells comprise naïve T cells,stem cell memory T cells, and/or central memory T cells. In someembodiments, the isolated population of immune cells treated with one ormore agents comprises NKT cells, as such the obtained one or moredesired subpopulation of immune cells comprise type I NKT cells. In yetsome other embodiments, the isolated population of immune cells treatedwith one or more agents comprises NK cells, as such the one or moredesired subpopulation of immune cells comprise adaptive NK cells.

In some embodiments of the composition as provided, the isolatedpopulation of immune cells may be isolated from peripheral blood, bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, or tumors of asubject. The subject may be healthy, may have an autoimmune disease, ahematopoietic malignancy, a virus infection or a solid tumor, or mayhave been previously administered with genetically modified immunecells. In some embodiments, the subject may be CMV seropositive. In someother embodiments, the isolated immune cells for modulation aregenetically modified (genetically engineered or naturally derived fromrearrangements, mutations, genetic imprinting and/or epigeneticmodification). In some embodiments, the isolated immune cells formodulation comprise at least one genetically modified modality. In someembodiments, the isolated population of immune cells are genomicallyengineered and comprise an insertion, a deletion, and/or a nucleic acidreplacement. In some particular embodiments, the immune cells comprisean exogenous nucleic acid encoding a T Cell Receptor (TCR), a ChimericAntigen Receptor (CAR), and/or overexpression of CD16 or a variantthereof. As such, the genetically modified immune cells are isolated forex vivo modulation using the present compositions and methods asdisclosed. In some embodiments, after modulation, the geneticallymodified immune cells isolated from a subject may be administered to thesame donor or a different patient.

In some embodiments of the composition as provided, the isolatedpopulation of immune cells may be differentiated from a stem cell, ahematopoietic stem or progenitor cell, or a progenitor cell. In someembodiments, the isolated population of immune cells may bedifferentiated from a stem cell, a hematopoietic stem or progenitorcell, or a progenitor cell prior to, or during, the treatment by theagent(s). In some embodiments, the stem cell is an induced pluripotentstem cell (iPSC) or embryonic stem cell (ESC). In some embodiments, theprogenitor cell is a CD34+ hemogenic endothelium cell, a multipotentprogenitor cell, a T cell progenitor cell, a NK progenitor cell, or aNKT progenitor cell. In some further embodiment, the stem cell,hematopoietic stem or progenitor cell, progenitor, the derived immunecell for modulation, or modulated derived immune cell is genomicallyengineered, for example, comprising an insertion, a deletion, and/or anucleic acid replacement. In one particular embodiment, the stem cell,hematopoietic stem or progenitor cell, or progenitor comprises anexogenous nucleic acid encoding a T Cell Receptor (TCR), a ChimericAntigen Receptor (CAR), and/or overexpression of CD16.

In some other embodiment of the composition as provided, the isolatedpopulation of immune cells may be trans-differentiated from anon-pluripotent cell of hematopoietic or non-hematopoietic lineage. Insome embodiments, the isolated population of immune cells may betrans-differentiated from a non-pluripotent cell of hematopoietic ornon-hematopoietic lineage prior to, or during, the treatment by theagent.

In some embodiments of the composition as provided, the isolatedpopulation of immune cells comprise T cells that have been modulatedwith a composition comprising an mTOR inhibitor. In some embodiments ofthe composition as provided, the isolated population of immune cellscomprise T cells that have been modulated with a composition comprisingan mTOR inhibitor, and dmPGE2 or an analogue or derivative of dmPGE2. Insome embodiments, the mTOR inhibitor is selected from rapamycin, andanalogues and derivatives thereof comprising sirolimus, sirolimusderivatives, temsirolimus, 40-O-(2-hydroxy)ethyl-rapamycin (everolimus),40-O-(3-hydroxy)propyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, 40-O-tetrazole-rapamycin, andother O-alkylated or O-methylated rapamycin derivatives. In someembodiments, the analogue or derivative of dmPGE2 is selected from thegroup consisting of PGE₂, 16,16-dimethyl PGE₂ p-(p-acetamidobenzamido)phenyl ester, 1 l-deoxy-16,16-dimethyl PGE₂, 9-deoxy-9-methylene-16,16-dimethyl PGE₂, 9-deoxy-9-methylene PGE₂, 9-keto Fluprostenol, 5-transPGE₂, 17-phenyl-omega-trinor PGE₂, PGE₂ serinol amide, PGE₂ methylester, 16-phenyl tetranor PGE₂, 15(S)-15-methyl PGE₂, 15(R)-15-methylPGE₂, 8-iso-15-keto PGE₂, 8-iso PGE2 isopropyl ester,8-iso-16-cyclohexyl-tetranor PGE₂, 20-hydroxy PGE₂, 20-ethyl PGE₂,11-deoxy PGEi, nocloprost, sulprostone, butaprost, 15-keto PGE₂, and 19(R) hydroxy PGE₂. In yet some other embodiments, the compositioncomprises rapamycin and dmPGE₂.

In some embodiments of the composition as provided, the isolatedpopulation of immune cells comprise T cells. In some embodiments, theisolated population of immune cells comprise CAR-T cells. In someembodiments, the modulated immune cells comprising T cells having atleast one of the following properties: (1) increased gene expression inat least one of CD27, CCR7, CD62L, TCF7, LEF1, BLIMP-1, ALDOC, and ENO2;(2) decreased gene expression in at least one of PD-1 and Tim-3; (3)increased spare respiratory capacity (SRC); (4) increased central memoryT cell subpopulation; (5) decreased effector T cell subpopulation; (6)improved expansion and viability; and (7) improved capability in tumorclearance and persistence, when compared to T cells without beingmodulated with a composition comprising an mTOR inhibitor, andoptionally dmPGE2 or an analogue or derivative of dmPGE2. In someembodiments, the T cells having at least one of the above properties areCAR-T cells.

Another aspects of the present invention provides a compositioncomprising an isolated population of T cells having at least one of theproperties: (1) increased gene expression in at least one of CD27, CCR7,CD62L, TCF7, LEF1, BLIMP-1, ALDOC, and ENO2; (2) decreased geneexpression in at least one of PD-1 and Tim-3; (3) increased sparerespiratory capacity (SRC); (4) increased central memory T cellsubpopulation; and (5) decreased effector T cell subpopulation, when incomparison to T cells without being modulated. In some embodiments, theisolated population of T cells in the composition comprise CAR-T cells.In some embodiments, the isolated population of T cells in thecomposition have improved capability in at least one of: expansion;viability; persistence and tumor clearance.

Another aspect of the present invention provides a method of modulatinga population of immune cells for adoptive therapies, the methodgenerally comprises contacting the population of immune cells with asufficient amount of a composition comprising at least one agentselected from the group consisting of the compounds listed in Table 1,and derivatives and analogues thereof, for a time sufficient to obtain apopulation of modulated immune cells having improved therapeuticpotential for adoptive cell therapy compared to unmodulated immunecells. In some embodiments, the modulated immune cells for adoptivetherapies are autologous. In some embodiments, the modulated immunecells for adoptive therapies are allogenic.

In some embodiments of the method, contacting the population of immunecells with the one or more agents improves proliferation, cytotoxicity,cytokine response, cytokine release, cell recall, and/or persistence;and/or improves cell expansion, maintenance, differentiation,de-differentiation, and/or survival rate in comparison to immune cellswithout the treatment by the one or more agents of Table 1, andderivative or analogs thereof. In some embodiments of the method,contacting the population of immune cells with one or more agents ofTable 1, and derivative or analogs thereof, increases the number orratio of one or more desired subpopulations of the immune cells incomparison to immune cells without the treatment by the same one or moreagents.

In some embodiments, the above method further comprises (b) isolatingthe one or more desired subpopulations of the immune cells contactedwith the one or more agents of Table 1.

In some embodiments, the above method further comprises administeringthe population or a subpopulation of the treated immune cells of step(a), or the isolated one or more desired subpopulations of the immunecells of step (b), or the therapeutical composition thereof to a subjectin need of cell therapy. In some embodiments, the subject has anautoimmune disorder, hematological malignancy, solid tumor, orinfection. In some embodiments, the subject had, is under, or will betreated with, chemotherapy or radiation therapy.

In some embodiments, the population of immune cells comprises T cells,NKT cells, or NK cells. In one embodiment of the method, the populationof immune cells comprises T cells, and the one or more desiredsubpopulations after treatment comprise naïve T cells, stem cell memoryT cells, and/or central memory T cells. In one embodiment of the method,the population of immune cells comprises NKT cells, and the one or moredesired subpopulations after treatment comprise type I NKT cells. In oneembodiment of the method, the population of immune cells comprises NKcells, and the one or more desired subpopulations after treatmentcomprise adaptive NK cells.

In some embodiments of said general method, the immune cells formodulation are isolated from or comprised in peripheral blood, bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, or tumors. Insome embodiments, the immune cells for modulation are isolated from ahealthy subject; a subject having an autoimmune disease, a hematopoieticmalignancy, a virus infection or a solid tumor; a subject previouslyadministered with genetically modified immune cells; or a subject thatis CMV seropositive. In some other embodiments, the isolated immunecells for modulation are genetically modified (genetically engineered ornaturally derived from rearrangements, mutations, genetic imprintingand/or epigenetic modification). In some embodiments, the isolatedimmune cells for modulation comprise at least one genetically modifiedmodality. In some embodiments, the isolated population of immune cellsare genomically engineered and comprise an insertion, a deletion, and/ora nucleic acid replacement. In some particular embodiments, the immunecells comprise an exogenous nucleic acid encoding a T Cell Receptor(TCR), a Chimeric Antigen Receptor (CAR), and/or overexpression of CD16or a variant thereof. As such, the genomically modified immune cells areisolated for ex vivo modulation using the present compositions andmethods as disclosed. In some embodiments, after modulation, thegenomically modified immune cells isolated from a subject may beadministered to the same donor or a different patient.

In some embodiments, the immune cells for modulation are differentiatedin vitro from stem cells, hematopoietic stem or progenitor cells, orprogenitor cells. In some embodiments, the immune cells for modulationare trans-differentiated in vitro from non-pluripotent cells ofhematopoietic or non-hematopoietic lineage. In some embodiments, saidstem cells comprise induced pluripotent stem cells (iPSCs) or embryonicstem cells (ESCs). In some embodiments, said progenitor cell is a CD34+hemogenic endothelium cell, a multipotent progenitor cell, a T cellprogenitor cell, a NK progenitor cell, or a NKT progenitor cell. In yetsome other embodiments, the stem cell, hematopoietic stem or progenitorcell, or progenitor cell is genomically engineered and comprises aninsertion, a deletion, or a nucleic acid replacement, and/or comprisesat least one genetically modified modality. As such, the desiredsubpopulation of modulated immune cells derived therefrom comprisesimmune cells having at least one genetically modified modality.

In some embodiments, said genetically modified modality comprises atleast one of safety switch proteins, targeting modalities, receptors,signaling molecules, transcription factors, pharmaceutically activeproteins and peptides, drug target candidates; or proteins promotingengraftment, trafficking, homing, viability, self-renewal, persistence,immune response regulation and modulation, and/or survival of the immunecells. In some other embodiments, the genetically modified modalitiescomprise one or more of deletion or reduced expression of B2M, TAP1,TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP,and any gene in the chromosome 6p21 region. In some other embodiments,the genetically modified modalities comprise one or more introduced orincreased expression of HLA-E, HLA-G, HACD16, hnCD16, 41BBL, CD3, CD4,CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A2AR, Fc receptor, orsurface triggering receptors for coupling with bi- or multi-specific oruniversal engagers.

In some embodiments of the method of modulating immune cells, said “timesufficient” or “sufficient length of time” is no less than 16 hours, 14hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour,0.5 hour, 0.1 hour, or any length of time in between. As such, saidsufficient length of time, for example, is no less than 15, 13, 11, 9,7, 5, 3, 1, 0.5, or 0.1 hour(s). In some other embodiments of themethod, said sufficient length of time is no less than 24 hours, 36hours, 48 hours, 60 hours, 72 hours, or any length of time in between.As such, said sufficient length of time is, for example, no less than30, 42, 54, 66, 78, 90 hour(s).

In some embodiments of said method, the immune cells, during and/orafter modulation, are in a feeder-free environment. Feeder-freeconditions include feeder cell free, and feeder-conditioned medium free.In some embodiments of said method, the immune cells, during modulation,are co-cultured with feeder cells.

In some embodiments, the subject can be a candidate for adoptive celltransfer. In some embodiments, the subject can be a candidate for bonemarrow or stem cell transplantation. In some embodiments, the subjecthas previously received a bone marrow or stem cell transplantation. Insome embodiments, the subject has received bone marrow ablative ornon-myeolablative chemotherapy or radiation therapy.

In some embodiments of method, the composition for contacting the immunecells comprise an mTOR inhibitor. In some embodiments of method, thecomposition for contacting the immune cells comprise an mTOR inhibitor,and dmPGE2 or an analogue or derivative of dmPGE2. In some embodiments,the mTOR inhibitor is selected from rapamycin, and analogues andderivatives thereof comprising sirolimus, sirolimus derivatives,temsirolimus, 40-O-(2-hydroxy)ethyl-rapamycin (everolimus),40-O-(3-hydroxy)propyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, 40-O-tetrazole-rapamycin, andother O-alkylated or O-methylated rapamycin derivatives. In someembodiments, the analogue or derivative of dmPGE2 is selected from thegroup consisting of PGE₂, 16,16-dimethyl PGE₂ p-(p-acetamidobenzamido)phenyl ester, 1 l-deoxy-16,16-dimethyl PGE₂, 9-deoxy-9-methylene-16,16-dimethyl PGE₂, 9-deoxy-9-methylene PGE₂, 9-keto Fluprostenol, 5-transPGE₂, 17-phenyl-omega-trinor PGE₂, PGE₂ serinol amide, PGE₂ methylester, 16-phenyl tetranor PGE₂, 15(S)-15-methyl PGE₂, 15(R)-15-methylPGE₂, 8-iso-15-keto PGE₂, 8-iso PGE2 isopropyl ester,8-iso-16-cyclohexyl-tetranor PGE₂, 20-hydroxy PGE₂, 20-ethyl PGE₂,11-deoxy PGEi, nocloprost, sulprostone, butaprost, 15-keto PGE₂, and 19(R) hydroxy PGE₂. In yet some other embodiments, the composition forcontacting the immune cells comprises rapamycin and dmPGE₂.

In some embodiments of the method as provided, the population of immunecells comprise T cells. In some embodiments, the population of immunecells comprise CAR-T cells. In some embodiments, the modulated cellpopulation comprising T cells having at least one of the followingproperties: (1) increased gene expression in at least one of CD27, CCR7,CD62L, TCF7, LEF1, BLIMP-1, ALDOC, and ENO2; (2) decreased geneexpression in at least one of PD-1 and Tim-3; (3) increased sparerespiratory capacity (SRC); (4) increased central memory T cellsubpopulation; (5) decreased effector T cell subpopulation; (6) improvedexpansion and viability; and (7) improved capability in tumor clearanceand persistence, when compared to T cells without being modulated with acomposition comprising an mTOR inhibitor, and dmPGE2 or an analogue orderivative of dmPGE2. In some embodiments, the T cells having at leastone of the above properties are CAR-T cells.

An additional aspect of the invention provides a method of making atherapeutic composition for cell therapies according to any of the abovemethods for modulating a population of immune cells.

A further aspect of the present invention provides using the aboveimmune cell modulation methods to make therapeutic compositionscomprising modulated immune cells for cell therapies. In someembodiment, the modulated immune cells comprise T, NK and/or NKT cells.In some embodiments, the modulated NK cells comprise adaptive NK cells.An additional aspect of the present invention provides a population ofmodulated immune cells comprising selectively expanded NK cells made bythe method provided herein.

Yet another aspect of the present invention provides a therapeuticcomposition comprising the modulated cells obtained using the methodsand composition disclosed herein, and a therapeutically acceptablemedium. In some embodiments of the therapeutic composition, thecomposition further comprises one of more additional additives selectedfrom the group consisting of peptides, cytokines, mitogens, growthfactors, small RNAs, dsRNAs (double stranded RNAs), mononuclear bloodcells, feeder cells, feeder cell components or replacement factors,vectors comprising one or more polynucleic acids of interest,antibodies, chemotherapeutic agents or radioactive moiety, andimmunomodulatory drugs (IMiDs).

Further provided is a method of treating a subject by administering atherapeutically sufficient amount of the above said therapeuticcomposition to a subject in need of an adoptive cell therapy. In someembodiments, the cell therapy is autologous. In some other embodiments,the cell therapy is allogeneic. In some embodiments, the subject in needof the therapy has an autoimmune disorder, a hematological malignancy, asolid tumor, cancer, or an infection associated with HIV, RSV, EBV, CMV,adenovirus, or BK polyomavirus. In some embodiments, the method oftreating a subject using the modulated immune cells is carried out byadministering said therapeutic composition in combination with anantibody, a chemotherapeutic, or a radioactive treatment, wherein theantibody, chemotherapeutic, or radioactive treatment is prior to, duringor after administering the therapeutic composition.

Yet another aspect of the invention provides use of a mixture formanufacturing of a therapeutic composition for cell therapies, whereinthe mixture comprises: (a) an isolated population of immune cells, and(b) a composition comprising an mTOR inhibitor, or a combination of anmTOR inhibitor, and dmPGE2 or an analogue or derivative of dmPGE2. Insome embodiments, the mTOR inhibitor is selected from rapamycin, andanalogues and derivatives thereof. In some embodiments, the analoguesand derivatives of rapamycin is selected from the group consisting ofsirolimus, sirolimus derivatives, temsirolimus,40-O-(2-hydroxy)ethyl-rapamycin (everolimus),40-O-(3-hydroxy)propyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, 40-O-tetrazole-rapamycin, orother O-alkylated or O-methylated rapamycin derivatives. In some otherembodiments, the analogue or derivative of dmPGE2 is selected from thegroup consisting of PGE₂, 16,16-dimethyl PGE₂ p-(p-acetamidobenzamido)phenyl ester, 11-deoxy-16,16-dimethyl PGE₂, 9-deoxy-9-methylene-16,16-dimethyl PGE₂, 9-deoxy-9-methylene PGE₂, 9-keto Fluprostenol, 5-transPGE₂, 17-phenyl-omega-trinor PGE₂, PGE₂ serinol amide, PGE₂ methylester, 16-phenyl tetranor PGE₂, 15(S)-15-methyl PGE₂, 15(R)-15-methylPGE₂, 8-iso-15-keto PGE₂, 8-iso PGE2 isopropyl ester,8-iso-16-cyclohexyl-tetranor PGE₂, 20-hydroxy PGE₂, 20-ethyl PGE₂,11-deoxy PGEi, nocloprost, sulprostone, butaprost, 15-keto PGE₂, and 19(R) hydroxy PGE₂. In one particular embodiment, the mixture formanufacturing a therapeutic composition for cell therapies comprises acombination of rapamycin and dmPGE2.

As provided, in the mixture for manufacturing of a therapeuticcomposition for cell therapies, the composition comprising a combinationof an mTOR inhibitor, and dmPGE2 or an analogue or derivative of dmPGE2is capable of modulating the isolated population of immune cells. Insome embodiments the mixture for manufacturing a therapeutic compositionfor cell therapies comprises an isolated population of T cells. In someembodiments, the T cells are CAR-T cells.

In some embodiments, after being modulated by the combination of an mTORinhibitor, and dmPGE2 or an analogue or derivative of dmPGE2, themodulated immune cells comprises T cells having at least one of thefollowing properties: (1) increased gene expression in at least one ofCD27, CCR7, CD62L, TCF7, LEF1, BLIMP-1, ALDOC, and ENO2; (2) decreasedgene expression in at least one of PD-1 and Tim-3; (3) increased sparerespiratory capacity (SRC); (4) increased central memory T cellsubpopulation; (5) decreased effector T cell subpopulation; (6) improvedexpansion and viability; and (7) improved capability in tumor clearanceand persistence, when compared to T cells without being modulated.

Various objects and advantages of this use will become apparent from thefollowing description taken in conjunction with the accompanyingdrawings wherein are set forth, by way of illustration and example,certain embodiments of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are graphic representations of z-Score of the percentage ofcells co-expressing both of CCR7 and CD62L, and a relative measure ofthe absolute number of naïve, stem cell memory, or central memory Tcells in (FIG. 1A) the viable CD8+ cell population and (FIG. 1B) theviable CD4+ cell population.

FIG. 2 shows the various CAR constructs used in the studies.

FIG. 3 shows CD27 surface expression on CAR-T cells treated under DMSO(vehicle), rapamycin, dmPGE2, and rapamycin+dmPGE2 combination,respectively.

FIG. 4 shows CAR-T cells treated with rapamycin+dmPGE2 combinationacquire a central memory phenotype. A: T cell subsets including centralmemory (Tcm), naïve (Tn), effector memory (Tem) and CD45RA⁺ effectormemory (Temra) T cells. B: T cell subsets present in cultures afterDMSO, rapamycin, dmPGE2, and rapamycin+dmPGE2 combination treatment. C:Flow cytometry analysis used to identify the different subsets underrespective compound treatment.

FIG. 5 shows the expression exhaustion markers, PD-1 and Tim-3, in CD8CAR-T cells treated under DMSO, rapamycin, dmPGE2, and rapamycin+dmPGE2combination.

FIG. 6 shows oxygen consumption rate (OCR) in cells treated under DMSO,rapamycin, dmPGE2, and rapamycin+dmPGE2 combination.

FIGS. 7A-7B show genome-wide expression characterization of CD8+ T-cellsunder different compound treatment. FIG. 7A: the comparison ofgenome-wide transcriptional profiles induced by rapamycin, dmPGE2, andrapamycin+dmPGE2 combination by probes included in the microarray. FIG.7B: Venn diagram representing the number of genes having differentialchanges (up or down regulated 2 fold or more in comparison to thevehicle treatment) under rapamycin, dmPGE2, and/or rapamycin+dmPGE2combination treatments.

FIG. 8 shows expression level comparison of genes relevant to T cellfunction, differentiation, and metabolism in T cells treated withrapamycin, dmPGE2, and rapamycin+dmPGE2 combination, respectively. A.Transcriptional changes on many T cell genes that are related to amemory phenotype as well metabolism. B. Transcriptional change of TCF7,LEF1, BLIMP-1 (or PRDM1). C. Transcriptional change of ALDOC, ENO andPGK1 genes under different treatment.

FIG. 9 shows RT-qPCR quantitation of memory cell marker genes in CD8+T-cells treated with treated with DMSO, rapamycin, dmPGE2, andrapamycin+dmPGE2 combination. A: CCR7 mRNA quantification. B: CD62L mRNAquantification. C: CD27 mRNA quantification.

FIG. 10 shows in vitro serial killing assay to assess CAR-T cellexpansion in the presence of tumor cells, and the CAR-T cells weretreated with DMSO, rapamycin, dmPGE2, and rapamycin+dmPGE2 combination.A: Fold expansion of CAR-T cells for each round of killing. B:Comparison of total fold expansion of treated CAR-T cells over 3 roundsof killing.

FIG. 11 shows the killing ability of the CAR-T cells in vitro wasunaffected in the three rounds of killing assays. A: CAR-T cellspreviously treated with rapa (abbreviation for “rapamycin”), dmPGE2, orrapa+dmPGE2 all cleared target cells over time. B: Flow cytometryanalysis of viable cells in the co-cultures show very few target cellsremaining under vehicle, rapamycin, dmPGE2, and rapamycin+dmPGE2combination.

FIG. 12 shows the effect of adding rapamycin and dmPGE2 in combinationto CD4 and CD8 T cell in larger-scale culture format at 1 week postactivation. A: increased expansion relative to DMSO. B: Increasedviability relative to DMSO.

FIGS. 13A-13B show improved in vivo efficacy of rapa+dmPGE2-treatedCAR-T cells. FIG. 13A: CAR-T cells treated with rapa+dmPGE2 or the PI3Kinhibitor PI-103 were able to clear tumor from the majority of mice,while those treated with untransduced T cells, or CAR-T cells treatedwith DMSO, U0126 or TWS119, demonstrated minimal tumor control. FIG.13B: At 21 days post challenge with secondary tumor 75% of therapa+dmPGE2-treated CAR-T mice and 60% of the PI103 CAR-T-treated micehad no detectable tumor.

FIG. 14 shows rapa+dmPGE2 treatment increased the in vivo tumorclearance and persistence of CAR-T cells after cryopreservation.

FIG. 15 shows tumor clearance and persistence of suboptimal doses ofCAR-T cells under (A) rapa and (B) rapa+dmPGE2 treatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods of modulatingimmune cell populations or subpopulations to obtain improved therapeuticpotential for adoptive immunotherapies. The present invention alsoprovides the method of using the modulated immune cells having improvedtherapeutic potential. In general, immune cells having improvedtherapeutic potential exhibit at least one of the following: improvedproliferation, persistence, cytotoxicity, and/or cell recall/memory. Theinvention provides methods of improving immune cell therapeuticpotential through improvements to the quality of the immune cells—forexample, an increase in the number or ratio of a subpopulation of cellsthat displays improvement in at least one of the following qualitieswould be expected to result in better immunotherapeutic results:migration, homing, cytotoxicity, maintenance, expansion, persistence,longevity, differentiation, and/or de-differentiation of the same cells.

Definition

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

As used herein, the articles “a,” “an,” and “the” refer to one or tomore than one of the grammatical object of the article. By way ofexample, a T cell means one T cell or more than one T cells.

As used herein, the terms “T lymphocyte” and “T cell” are usedinterchangeably and refer to a principal type of white blood cell thatcompletes maturation in the thymus and that has various roles in theimmune system, including the identification of specific foreign antigensin the body and the activation and deactivation of other immune cells. AT cell can be any T cell, such as a cultured T cell, e.g., a primary Tcell, or a T cell from a cultured T cell line, e.g., Jurkat, SupT1,etc., or a T cell obtained from a mammal. The T cell can be CD3+ cells.The T cell can be any type of T cell and can be of any developmentalstage, including but not limited to, CD4+/CD8+ double positive T cells,CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g.,cytotoxic T cells), peripheral blood mononuclear cells (PBMCs),peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes(TILs), memory T cells, naïve T cells, regulator T cells, gamma delta Tcells (γδ T cells), and the like. Additional types of helper T cellsinclude cells such as Th3, Th17, Th9, or Tfh cells. Additional types ofmemory T cells include cells such as central memory T cells (Tcm cells),effector memory T cells (Tem cells and TEMRA cells). The T cell can alsorefer to a genetically engineered T cell, such as a T cell modified toexpress a T cell receptor (TCR) or a chimeric antigen receptor (CAR).The T cell can also be differentiated from a stem cell or progenitorcell.

As used herein, the term “naïve T cell” or Tn, refers to mature T cellsthat, unlike activated or memory T cells, have not encountered theircognate antigen within the periphery. Naïve T cells are commonlycharacterized by the surface expression of L-selectin (CD62L); theabsence of the activation markers CD25, CD44 or CD69; and the absence ofthe memory CD45RO isoform. They also express functional IL-7 receptors,consisting of subunits IL-7 receptor-α, CD127, and common-7 chain,CD132. In the naïve state, T cells are thought to be quiescent andnon-dividing, requiring the common-gamma chain cytokines IL-7 and IL-15for homeostatic survival mechanisms.

As used herein, the term “central memory T cells” or Tcm, refers to asubgroup or subpopulation of T cells that have lower expression orpro-apoptotic signaling genes, for example, Bid, Bnip3 and Bad, and havehigher expression of genes associated with trafficking to secondarylymphoid organs, which genes include CD62L, CXCR3, CCR7, in comparisonto effector memory T cells, or Tem.

As used herein, the term “stem memory T cells,” or “stem cell memory Tcells”, or Tscm, refers to a subgroup or subpopulation of T cells thatare capable of self-renewing and generating Tcm, Tem and Teff (effectorT cells), and express CD27 and lymphoid homing molecules such as CCR7and CD62L, which are properties important for mediating long-termimmunity. As used herein, the term “NK cell” or “Natural Killer cell”refer to a subset of peripheral blood lymphocytes defined by theexpression of CD56 or CD16 and the absence of the T cell receptor (CD3).As used herein, the terms “adaptive NK cell” and “memory NK cell” areinterchangeable and refer to a subset of NK cells that arephenotypically CD3- and CD56+, expressing and have at least one ofCD57+, NKG2C and CD57, and optionally, CD16, but lack expression of oneor more of the following: +, low PLZF, low SYK, FceR

, and low FεFRγ, low EAT-2., low TIGIT, low PD1, low CD7, low CD161,high LILRB1, high CD45RO, and low CD45RA. In some embodiments, isolatedsubpopulations of CD56+NK cells comprise expression of NKG2C and CD57.In some other embodiments, isolated subpopulations of CD56+NK cellscomprise expression of CD57, CD16, NKG2C, CD57, NKG2D, NCR ligands,NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A and/orDNAM-1. CD56+ can be dim or bright expression.

As used herein, the term “NKT cells” or “natural killer T cells” refersto CDld-restricted T cells, which express a T cell receptor (TCR).Unlike conventional T cells that detect peptide antigens presented byconventional major histocompatibility (MHC) molecules, NKT cellsrecognize lipid antigens presented by CD1d, a non-classical MHCmolecule. Two types of NKT cells are currently recognized. Invariant ortype I NKT cells express a very limited TCR repertoire—a canonicalα-chain (Vα24-Jα18 in humans) associated with a limited spectrum of βchains (Vβ11 in humans). The second population of NKT cells, callednonclassical or noninvariant type II NKT cells, display a moreheterogeneous TCR αβ usage. Type I NKT cells are currently consideredsuitable for immunotherapy. Adaptive or invariant (type I) NKT cells canbe identified with the expression of at least one or more of thefollowing markers, TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer,CD161 and CD56.

As used herein, the term “isolated” or the like refers to a cell, or apopulation of cells, which has been separated from its originalenvironment, i.e., the environment of the isolated cells issubstantially free of at least one component as found in the environmentin which the “un-isolated” reference cells exist. The term includes acell that is removed from some or all components as it is found in itsnatural environment, for example, tissue, biopsy. The term also includesa cell that is removed from at least one, some or all components as thecell is found in non-naturally occurring environments, for example,culture, cell suspension. Therefore, an isolated cell is partly orcompletely separated from at least one component, including othersubstances, cells or cell populations, as it is found in nature or as itis grown, stored or subsisted in non-naturally occurring environments.Specific examples of isolated cells include partially pure cells,substantially pure cells and cells cultured in a medium that isnon-naturally occurring. Isolated cells may be obtained from separatingthe desired cells, or populations thereof, from other substances orcells in the environment, or from removing one or more other cellpopulations or subpopulations from the environment. As used herein, theterm “purify” or the like refers to increasing purity. For example, thepurity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%,or 100%.

As used herein, the term “population” when used with reference to T, NKor NKT cells refers to a group of cells including two or more T, NK, orNKT cells, respectively. Using T cell as an example, the isolated, orenriched, population of T cells may include only one type of T cell, ormay include a mixture of two or more types of T cell. The isolatedpopulation of T cells can be a homogeneous population of one type of Tcell or a heterogeneous population of two or more types of T cell. Theisolated population of T cells can also be a heterogeneous populationhaving T cells and at least a cell other than a T cell, e.g., a B cell,a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelialcell, an epithelial cell, a muscle cell, a brain cell, etc. Theheterogeneous population can have from 0.01% to about 100% T cell.Accordingly, an isolated population of T cells can have at least 50%,60%, 70%, 80%, 90%, 95%, 98%, or 99% T cells. The isolated population ofT cells can include one or more, or all of, the different types of Tcells, including but not limited to those disclosed herein. In anisolated population of T cells that includes more than one type of Tcells, the ratio of each type of T cell can range from 0.01% to 99.99%.The isolated population also can be a clonal population of T cells, inwhich all the T cells of the population are clones of a single T cell.

An isolated population of T, NK or NKT cells may be obtained from anatural source, such as human peripheral blood or cord blood. Variousways of dissociating cells from tissues or cell mixtures to separate thevarious cell types have been developed in the art. In some cases, thesemanipulations result in a relatively homogeneous population of cells.The T cells can be isolated by a sorting or selection process asdescribed herein or by other methods known in the art. The proportion ofT cells in the isolated population may be higher than the proportion ofT cells in the natural source by at least about 10%, about 20%, about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%,about 90%, or about 95%. The isolated population of T cells can be for Tcells in general, or one or more specific types of T cells.

As used herein, the term “subpopulation” when used in reference to T, NKor NKT cells refers to a population of T, NK or NKT cells that includesless than all types of T, NK, or NKT cells, respectively, that are foundin nature.

As used herein, the term “pluripotent” refers to the ability of a cellto form all lineages of the body or soma (i.e., the embryo proper). Forexample, embryonic stem cells are a type of pluripotent stem cells thatare able to form cells from each of the three germs layers, theectoderm, the mesoderm, and the endoderm. Pluripotency is a continuum ofdevelopmental potencies ranging from the incompletely or partiallypluripotent cell (e.g., an epiblast stem cell or EpiSC), which is unableto give rise to a complete organism to the more primitive, morepluripotent cell, which is able to give rise to a complete organism(e.g., an embryonic stem cell).

As used herein, the term “induced pluripotent stem cells” or, “iPSCs,”refers to stem cells produced from differentiated adult cells that havebeen induced or changed (i.e. reprogrammed) into cells capable ofdifferentiating into tissues of all three germ or dermal layers:mesoderm, endoderm, and ectoderm.

As used herein, the term “embryonic stem cell” refers to naturallyoccurring pluripotent stem cells of the inner cell mass of the embryonicblastocyst. Embryonic stem cells are pluripotent and give rise duringdevelopment to all derivatives of the three primary germ layers:ectoderm, endoderm and mesoderm. They do not contribute to theextra-embryonic membranes or the placenta and are not totipotent.

As used herein, the term “progenitor cell” refers to cells that havegreater developmental potential, i.e., a cellular phenotype that is moreprimitive (e.g., is at an earlier step along a developmental pathway orprogression) relative to a cell which it can give rise to bydifferentiation. Often, progenitor cells have significant or very highproliferative potential. Progenitor cells can give rise to multipledistinct cells having lower developmental potential, i.e.,differentiated cell types, or to a single differentiated cell type,depending on the developmental pathway and on the environment in whichthe cells develop and differentiate.

As used herein, the terms “reprogramming” or “dedifferentiation” or“increasing cell potency” or “increasing developmental potency” refersto a method of increasing the potency of a cell or dedifferentiating thecell to a less differentiated state. For example, a cell that has anincreased cell potency has more developmental plasticity (i.e., candifferentiate into more cell types) compared to the same cell in thenon-reprogrammed state. In other words, a reprogrammed cell is one thatis in a less differentiated state than the same cell in anon-reprogrammed state.

As used herein, the term “differentiation” is the process by which anunspecialized (“uncommitted”) or less specialized cell acquires thefeatures of a specialized cell such as, for example, a blood cell or amuscle cell. A differentiated or differentiation-induced cell is onethat has taken on a more specialized (“committed”) position within thelineage of a cell. The term “committed”, when applied to the process ofdifferentiation, refers to a cell that has proceeded in thedifferentiation pathway to a point where, under normal circumstances, itwill continue to differentiate into a specific cell type or subset ofcell types, and cannot, under normal circumstances, differentiate into adifferent cell type or revert to a less differentiated cell type.

As used herein, the term “encoding” refers to the inherent property ofspecific sequences of nucleotides in a polynucleotide, such as a gene, acDNA, or a mRNA, to serve as templates for synthesis of other polymersand macromolecules in biological processes having either a definedsequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a definedsequence of amino acids and the biological properties resultingtherefrom. Thus, a gene encodes a protein if transcription andtranslation of mRNA corresponding to that gene produces the protein in acell or other biological system. Both the coding strand, the nucleotidesequence of which is identical to the mRNA sequence and is usuallyprovided in sequence listings, and the non-coding strand, used as thetemplate for transcription of a gene or cDNA, can be referred to asencoding the protein or other product of that gene or cDNA.

As used herein, the term “exogenous” in intended to mean that thereferenced molecule or the referenced activity is introduced into thehost cell. The molecule can be introduced, for example, by introductionof an encoding nucleic acid into the host genetic material such as byintegration into a host chromosome or as non-chromosomal geneticmaterial such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe cell. The term “endogenous” refers to a referenced molecule oractivity that is present in the host cell. Similarly, the term when usedin reference to expression of an encoding nucleic acid refers toexpression of an encoding nucleic acid contained within the cell and notexogenously introduced.

As used herein, the term “polynucleotide” refers to a polymeric form ofnucleotides of any length, either deoxyribonucleotides orribonucleotides or analogs thereof. The sequence of a polynucleotide iscomposed of four nucleotide bases: adenine (A); cytosine (C); guanine(G); thymine (T); and uracil (U) for thymine when the polynucleotide isRNA. A polynucleotide can include a gene or gene fragment (for example,a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA),transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probesand primers. Polynucleotide also refers to both double- andsingle-stranded molecules.

As used herein, the term “peptide,” “polypeptide,” and “protein” areused interchangeably and refer to a molecule having amino acid residuescovalently linked by peptide bonds. A polypeptide must contain at leasttwo amino acids, and no limitation is placed on the maximum number ofamino acids of a polypeptide. As used herein, the terms refer to bothshort chains, which are also commonly referred to in the art aspeptides, oligopeptides and oligomers, for example, and to longerchains, which generally are referred to in the art as polypeptides orproteins. “Polypeptides” include, for example, biologically activefragments, substantially homologous polypeptides, oligopeptides,homodimers, heterodimers, variants of polypeptides, modifiedpolypeptides, derivatives, analogs, fusion proteins, among others. Thepolypeptides include natural polypeptides, recombinant polypeptides,synthetic polypeptides, or a combination thereof.

As used herein, the term “ex vivo” refers to activities that take placeoutside an organism, such as experimentation or measurements done in oron living tissue in an artificial environment outside the organism,preferably with minimum alteration of the natural conditions. The “exvivo” procedures can involve living cells or tissues taken from anorganism and cultured in a laboratory apparatus, usually under sterileconditions, and typically for a few hours or up to about 24 hours, butincluding up to 2 to 28 days, depending on the circumstances. Suchtissues or cells can also be collected and frozen, and later thawed forex vivo treatment. Tissue culture experiments or procedures lastinglonger than a few days using living cells or tissue are typicallyconsidered to be “in vitro,” though in certain embodiments, this termcan be used interchangeably with ex vivo. Meanwhile, an “in vivo”activity takes place inside an organism, such as cell engraftment, cellhoming, self-renewal of cells, and expansion of cells.

As used herein, the term “in vitro” refers to activities performed ortaking place in a test tube, culture dish, or elsewhere outside a livingorganism.

As used herein, the terms “agent,” “modulating agent,” and “modulator”are used interchangeably herein to refer to a compound or moleculecapable of modifying gene expression profile or a biological property ofa cell including an immune cell. The agent can be a single compound ormolecule, or a combination of more than one compound or molecule.

As used herein, the terms “contact,” “treat,” or “modulate,” when usedin reference to an immune cell, are used interchangeably herein to referto culturing, incubating or exposing an immune cell with one or more ofthe agents disclosed herein.

As used herein, a “noncontacted” or an “untreated” cell is a cell thathas not been treated, e.g., cultured, contacted, or incubated with anagent other than a control agent. Cells contacted with a control agent,such as DMSO, or contacted with another vehicle are examples ofnoncontacted cells.

As used herein, “feeder cells” or “feeders” are terms describing cellsof one type that are co-cultured with cells of a second type to providean environment in which the cells of the second type can grow, as thefeeder cells provide stimulation, growth factors and nutrients for thesupport of the second cell type. The feeder cells are optionally from adifferent species as the cells they are supporting. For example, certaintypes of human cells, including stem cells, can be supported by primarycultures of mouse embryonic fibroblasts, or immortalized mouse embryonicfibroblasts. In another example, peripheral blood derived cells ortransformed leukemia cells support the expansion and maturation ofnatural killer cells. The feeder cells may typically be inactivated whenbeing co-cultured with other cells by irradiation or treatment with ananti-mitotic agent such as mitomycin to prevent them from outgrowing thecells they are supporting. Feeder cells may include endothelial cells,stromal cells (for example, epithelial cells or fibroblasts), andleukemic cells. Without limiting the foregoing, one specific feeder celltype may be a human feeder, such as a human skin fibroblast. Anotherfeeder cell type may be mouse embryonic fibroblasts (MEF). In general,various feeder cells can be used in part to maintain pluripotency,direct differentiation towards a certain lineage, enhance proliferationcapacity and promote maturation to a specialized cell types, such as aneffector cell.

As used herein, a “feeder-free” (FF) environment refers to anenvironment such as a culture condition, cell culture or culture mediawhich is essentially free of feeder or stromal cells, and/or which hasnot been pre-conditioned by the cultivation of feeder cells.“Pre-conditioned” medium refers to a medium harvested after feeder cellshave been cultivated within the medium for a period of time, such as forat least one day. Pre-conditioned medium contains many mediatorsubstances, including growth factors and cytokines secreted by thefeeder cells cultivated in the medium. In some embodiments, afeeder-free environment is free of both feeder or stromal cells and isalso not pre-conditioned by the cultivation of feeder cells.

As used herein, the term “analogue” refers to a chemical molecule thatis similar to another chemical substance in structure and function,differing structurally by one single element or group, or more than onegroup (e.g., 2, 3, or 4 groups) if it retains the same chemical scaffoldand function as the parental chemical. Such modifications are routine topersons skilled in the art, and include, for example, additional orsubstituted chemical moieties, such as esters or amides of an acid,protecting groups such as a benzyl group for an alcohol or thiol, andtert-butoxylcarbonyl groups for an amine. Also included aremodifications to alkyl side chains, such as alkyl substitutions (e.g.,methyl, dimethyl, ethyl, etc.), modifications to the level of saturationor unsaturation of side chains, and the addition of modified groups suchas substituted phenyl and phenoxy. Analogues can also includeconjugates, such as biotin or avidin moieties, enzymes such ashorseradish peroxidase and the like, and including radio-labeled,bioluminescent, chemoluminescent, or fluorescent moieties. Also,moieties can be added to the agents described herein to alter theirpharmacokinetic properties, such as to increase half-life in vivo or exvivo, or to increase their cell penetration properties, among otherdesirable properties. Also included are prodrugs, which are known toenhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.).

As used herein, the term “increase” refers to the ability of an agent toproduce or cause a greater physiological response (i.e., downstreameffects) in a cell, as compared to the response caused by either vehicleor a control molecule/composition, e.g., increased production ofinterleukin 4 or interleukin 10 by an isolated population of T cells.The increase can be an increase in gene expression as a result ofincreased signaling through certain cell signaling pathways. An“increased” amount is typically a statistically significant amount, andcan include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including allintegers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7,1.8, etc.) compared to the response produced by vehicle (the absence ofan agent) or a control composition.

As used herein, the term “decrease” refers to the ability of an agent toproduce or cause a lesser physiological response (i.e., downstreameffects) in a cell, as compared to the response caused by either vehicleor a control molecule/composition. The decrease can be a decrease ingene expression, a decrease in cell signaling, or a decrease in cellproliferation. An “decreased” amount is typically a “statisticallysignificant” amount, and can include a decrease that is 1.1, 1.2, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000times) (including all integers and decimal points in between and above1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) the response produced by vehicle (theabsence of an agent) or a control composition.

As used herein, the term “synergy” or “synergistic” refers to acombination of two or more entities for an enhanced effect such that theworking together of the two or more entities produces an effect greaterthan the sum of their individual effects, as compared to “antagonistic,”which is used when two or more entities in a combination counteract orneutralize each other's effect; and compared to “additive,” which isused when two or more entities in a combination produce an effect nearlyequal to the sum of their individual effects.

As used herein, the terms “substantially free of,” when used to describea composition, such as a cell population or culture media, refers to acomposition that is free of a specified substance of any source, suchas, 95% free, 96% free, 97% free, 98% free, 99% free of the specifiedsubstance, or is undetectable as measured by conventional means. Similarmeaning can be applied to the term “absence of,” where referring to theabsence of a particular substance or component of a composition.

As used herein, the term “about” or “approximately” refers to aquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number,frequency, percentage, dimension, size, amount, weight or length. Therange of quantity, level, value, number, frequency, percentage,dimension, size, amount, weight or length can be ±15%, ±10%, ±9%, 8%,±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% about a reference quantity, level,value, number, frequency, percentage, dimension, size, amount, weight orlength.

As used herein, the term “subject,” refers to a mammal. A subject can bea human or a non-human mammal such as a dog, cat, bovid, equine, mouse,rat, rabbit, or transgenic species thereof.

As used herein, the term “treat,” and the like, when used in referenceto a subject, refer to obtaining a desired pharmacologic and/orphysiologic effect, including without limitation achieving animprovement or elimination of the symptoms of a disease. The effect canbe prophylactic in terms of completely or partially preventing a diseaseor symptom thereof and/or can be therapeutic in terms of achieving animprovement or elimination of symptoms, or providing a partial orcomplete cure for a disease and/or adverse effect attributable to thedisease. The term “treatment” includes any treatment of a disease in amammal, particularly in a human, and includes: (a) preventing thedisease from occurring in a subject which can be predisposed to thedisease but has not yet been diagnosed as having it; (b) inhibiting thedisease, or arresting its development; (c) relieving the disease, orcausing regression of the disease, or to completely or partiallyeliminate symptoms of the disease; and (d) restoring the individual to apre-disease state, such as reconstituting the hematopoietic system.

As used herein, “genetic modification” refers to genetic editingincluding those (1) naturally derived from rearrangements, mutations,genetic imprinting and/or epigenetic modification, (2) or obtainedthrough genomic engineering through insertion, deletion or substitutionin the genome of a cell. Genetic modification, as used herein, alsoincludes one or more retainable therapeutic attributes of a sourcespecific immune cell that is donor-, disease-, or treatmentresponse-specific,

As used herein, the term “genetic imprint” refers to genetic orepigenetic information that contributes to preferential therapeuticattributes in a source cell. In the aspect of a source cell obtainedfrom a specifically selected donor, disease or treatment context, thegenetic imprint contributing to preferential therapeutic attributes mayinclude any context specific genetic or epigenetic modifications whichmanifest a retainable phenotype, i.e. a preferential therapeuticattribute, irrespective of the underlying molecular events beingidentified or not. Donor-, disease-, or treatment response-specificsource cells may comprise genetic imprints that are retainable in iPSCsand derived hematopoietic lineage cells, which genetic imprints includebut are not limited to, prearranged monospecific TCR, for example, froma viral specific T cell or invariant natural killer T (iNKT) cell;trackable and desirable genetic polymorphisms, for example, homozygousfor a point mutation that encodes for the high-affinity CD16 receptor inselected donors; and predetermined HLA requirements, i.e., selectedHLA-matched donor cells exhibiting a haplotype with increasedpopulation. As used herein, preferential therapeutic attributes includeimproved engraftment, trafficking, homing, viability, self-renewal,persistence, immune response regulation and modulation, survival, andcytotoxicity of a derived cell. A preferential therapeutic attribute mayalso relate to antigen targeting receptor expression; HLA presentationor lack thereof, resistance to tumor microenvironment; induction ofbystander immune cells and immune modulations; improved on-targetspecificity with reduced off-tumor effect; resistance to treatment suchas chemotherapy.

As used herein, the term “safety switch protein” refers to an engineeredprotein designed to prevent potential toxicity or otherwise adverseeffects of a cell therapy. In some instances, the safety switch proteinexpression is conditionally controlled to address safety concerns fortransplanted engineered cells that have permanently incorporated thegene encoding the safety switch protein into its genome. Thisconditional regulation could be variable and might include controlthrough a small molecule-mediated post-translational activation andtissue-specific and/or temporal transcriptional regulation. The safetyswitch could mediate induction of apoptosis, inhibition of proteinsynthesis, DNA replication, growth arrest, transcriptional andpost-transcriptional genetic regulation and/or antibody-mediateddepletion. In some instance, the safety switch protein is activated byan exogenous molecule, e.g. a prodrug, that when activated, triggersapoptosis and/or cell death of a therapeutic cell. Examples of safetyswitch proteins, include, but are not limited to suicide genes such ascaspase 9 (or caspase 3 or 7), thymidine kinase, cytosine deaminase,B-cell CD20, modified EGFR, and any combination thereof. In thisstrategy, a prodrug that is administered in the event of an adverseevent is activated by the suicide-gene product and kills the transducedcell.

A “therapeutically sufficient amount”, as used herein, includes withinits meaning a non-toxic but sufficient and/or effective amount of theparticular therapeutic and/or pharmaceutical composition to which it isreferring to provide a desired therapeutic effect. The exact amountrequired will vary from subject to subject depending on factors such asthe patient's general health, the patient's age and the stage andseverity of the condition. In particular embodiments, a therapeuticallysufficient amount is sufficient and/or effective to ameliorate, reduce,and/or improve at least one symptom associated with a disease orcondition of the subject being treated.

I. Agents for Improving Efficacy of Cell-Based Adoptive Immunotherapy

The present invention provides a composition comprising one or moreagents in an amount sufficient for improving therapeutic potential ofimmune cells suitable for adoptive cell-based therapies. Immune cellshaving improved therapeutic potential present improved proliferation,persistence, cytotoxicity, and/or cell recall/memory. Immune cells mayhave specifically improved in vivo proliferation, in vivo persistence,in vivo cytotoxicity, and/or in vivo cell recall/memory. To improveimmune cell therapeutic potential generally requires better quality ofthe immune cells—in a T cell population, for example, increased numberor ratio of naïve T cell, stem cell memory T cell, and/or central memoryT cell through maintenance, expansion, differentiation, and/orde-differentiation thereof are indicative of better quality of the Tcells for improved in vivo adoptive therapeutic potential. In a NK cellpopulation, for example, increased number or ratio of adaptive NK cellsthrough maintenance, subtype skewing, expansion, differentiation, and/orde-differentiation thereof are indicative of better quality of the NKcells for improved in vivo adoptive therapeutic potential. With respectto a NKT cell population, for example, an increased number or ratio oftype I NKT cells through maintenance, subtype switching, expansion,differentiation, and/or de-differentiation thereof are indicative ofbetter quality of the NKT cells for improved in vivo adoptivetherapeutic potential.

The immune cells suitable for adoptive cell-based therapies arecontacted, treated, or modulated with one or more agents included inTable 1. The treatment with the agent(s) can modify the biologicalproperties of the cells, or a subpopulation of the cells, including bymodulating cell expansion, maintenance, differentiation,dedifferentiation, and/or survival rate, and/or increasingproliferation, cytotoxicity, persistence, and/or cell recall/memory, andthus the therapeutic potential of the cells treated. For example, thetreatment can improve the therapeutic immune cell survival rate both invitro and in vivo. Further, the treatment can alter the ratios ofdifferent subpopulation of the treated cell population. For example, inone embodiment, the number and proportion of naïve T cells, stem cellmemory T cells, and/or central memory T cells increase in an isolated Tcell population upon treatment using one or more of the agents selectedfrom Table 1, and derivatives and analogs thereof. In anotherembodiment, upon treatment of a NK cell population using one or more ofthe agents selected from Table 1, and derivatives and analogs thereof,the number and percentage of adaptive NK cells are increased in thepopulation.

TABLE 1 Agents for Immune Cell Modulation in Adoptive Cell TherapiesCompounds CAS Number Group Group Descriptor Dorsomorphin 866405-64-3 IMetabolism & Nutrient Sensing Heptelidic 74310-84-2 I Metabolism & acidNutrient Sensing 1-Pyrrolidine- 5108-96-3 I Metabolism & carbodithioicNutrient Sensing acid, ammonium salt 2- 154-17-6 I Metabolism &dexoyglucose Nutrient Sensing (2-DG) GSK3 Including for II SignalingPathways Inhibitor example-BIO: 667463-62-9; TWS119: 601514-19-6;CHIR99021: 252917-06-9 Rho kinase Including for II Signaling Pathwaysinhibitors example- Thiazovivin: 1226056-71-8 MEK Including for IISignaling Pathways inhibitors example- PD0325901: 391210-10-9; U0126:109511-58-2 PDK1 agonist Including for II Signaling Pathways example-PS48: 1180676-32-7 TGFβ Including for II Signaling Pathways inhibitorsexample- SB431542: 301836-41-9 6-Mer- 6112-76-1 II Signaling Pathwayscaptopurine AC-93253 108527-83-9 II Signaling Pathways iodide Tiratricol51-24-1 II Signaling Pathways PI-103 371935-74-9 II Signaling PathwaysFulvestrant 129453-61-8 II Signaling Pathways Thapsigargin 67526-95-8 IISignaling Pathways SU 4312 5812-07-7 II Signaling Pathways Telmisartan144701-48-4 II Signaling Pathways Cyclosporin A 59865-13-3 II SignalingPathways 1,3,5-tris(4- 263717-53-9 II Signaling Pathways hydroxy-phenyl)- 4-propyl-1H- pyrazole BAY 61-3606 732983-37-8 II SignalingPathways Protoporphyrin 553-12-8 II Signaling Pathways IX disodium mTORIncluding for II Signaling Pathways inhibitor example- Rapamycin:53123-88-9 HS173 1276110-06-5 II Signaling Pathways LY294002 154447-36-6II Signaling Pathways Pictilisib 957054-30-7 II Signaling Pathways5-Azacytidine 320-67-2 III Proliferation and Apoptosis Fludarabine21679-14-1 III Proliferation and Apoptosis Roscovitine, 186692-45-5 IIIProliferation and Apoptosis (S)-Isomer PAC-1 315183-21-2 IIIProliferation and Apoptosis 8-Quinolinol, 773-76-2 IV Anti-infective5,7-dichloro- Nitrofurantoin 67-20-9 IV Anti-infective 8-Quinolinol,130-26-7 IV Anti-infective 5-chloro- 7-iodo- 2- 64-73-3 IVAnti-infective Naphthacene- carboxamide, 7-chloro-4- (dimethyl- amino)-1,4,4a,5,5a,6, 11,12a-octahy Nifuroxazide 965-52-6 IV Anti-infectiveTosufloxacin 100490-36-6 IV Anti-infective hydrochloride Sertraline79617-96-2 V Other Diethylene- 67-43-6 V Other triamine- pentaaceticacid, penta sodium Edrophonium 116-38-1 V Other chloride BIX012941392399-03-9 V Other Terfenadine 50679-08-8 V Other dmPGE2 (16,39746-25-3 V Other 16-dimethyl Prostaglandin E2)

Without being limited by the theory, the agents of Table 1 improve thetherapeutic potential of an immune cell for adoptive therapy bymodulating cell expansion, metabolism, and/or cell differentiation viaregulating cell metabolism, nutrient sensing, proliferation, apoptosis,signal transduction, properties relating to infective process, and/orother aspects of cell function. As understood by those skilled in theart, the scope of the present invention also includes analogues orderivatives, including but not limited to, salt, ester, ether, solvate,hydrate, stereoisomer or prodrug of the listed agents in Table 1. Forexample, illustrative examples of analogues and derivatives of a Table 1agent, dmPGE₂ (16,16-dimethyl Prostaglandin E2), include, withoutlimitation, PGE₂, 16,16-dimethyl PGE₂ p-(p-acetamidobenzamido) phenylester, 11-deoxy-16,16-dimethyl PGE₂, 9-deoxy-9-methylene-16, 16-dimethylPGE₂, 9-deoxy-9-methylene PGE₂, 9-keto Fluprostenol, 5-trans PGE₂,17-phenyl-omega-trinor PGE₂, PGE₂ serinol amide, PGE₂ methyl ester,16-phenyl tetranor PGE₂, 15(S)-15-methyl PGE₂, 15(R)-15-methyl PGE₂,8-iso-15-keto PGE₂, 8-iso PGE2 isopropyl ester,8-iso-16-cyclohexyl-tetranor PGE₂, 20-hydroxy PGE₂, 20-ethyl PGE₂,11-deoxy PGEi, nocloprost, sulprostone, butaprost, 15-keto PGE₂, and 19(R) hydroxy PGE₂. Also included are PG analogues or derivatives having asimilar structure to PGE₂ that are substituted with halogen at the9-position (see, e.g., WO 2001/12596, the disclosure of which is herebyincorporated by reference in its entirety), as well as2-decarboxy-2-phosphinico prostaglandin derivatives, such as thosedescribed in U.S. Publication No. 2006/0247214, the disclosure of whichis hereby incorporated by reference in its entirety).

GSK3 (Glycogen synthase kinase 3) inhibitors can include antibodies thatbind, dominant negative variants of, and siRNA, microRNA, antisensenucleic acids, and other polynucleotides that target GSK3. Suitable GSK3inhibitor (GSK3i) for use in compositions contemplated herein include,but are not limited to: Kenpaullone, 1-Azakenpaullone, CHIR99021,CHIR98014, AR-A014418, CT 99021, CT 20026, SB216763, AR-A014418,lithium, TDZD-8, BIO, BIO-Acetoxime,(5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine,Pyridocarbazole-cyclopenadienylruthenium complex, TDZD-84-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione,2-Thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole, OTDZT,alpha-4-Dibromoacetophenone, AR-AO 144-18,3-(1-(3-Hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione;TWS119, L803 H-KEAPPAPPQSpP-NH2 or its myristoylated form;2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone; GF109203X; RG318220;TDZD-8; TIBPO; and OTDZT. In one embodiment, the GSK-3 inhibitor isCHIR99021, BIO, TWS119, or Kenpaullone. In one embodiment, the GSK3inhibitor is TWS119. In another embodiment, the GSK-3 inhibitor isCHIR99021. In yet another embodiment the GSK3 inhibitor is BIO.

MEK/ERK pathway inhibitors refer to inhibitors of either MEK or ERKserine/threonine kinases that are part of the Raf/MEK/ERK pathway.ERK/MEK inhibitors suitable for use in compositions contemplated hereininclude, but not limited to: PD0325901, PD98059, U0126, SL327, ARRY-162,PD184161, PD184352, sunitinib, sorafenib, vandetanib, pazopanib,axitinib, GSK1 120212, ARRY-438162, RGO126766, XL518, AZD8330, RDEA1 19,AZD6244, FR180204, PTK787,6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazol-e-5-carboxylicacid (2,3-dihydroxy-propoxy)-amide;6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-pyran-2-ylm-ethyl)-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide,1-[6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimida-zol-5-yl]-2-hydroxy-ethanone,6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazol-e-5-carboxylicacid (2-hydroxy-1,1-dimethyl-ethoxy)-amide,6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-furan-2-ylm-ethyl)-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide,6-(4-Bromo-2-fluoro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazol-e-5-carboxylicacid (2-hydroxy-ethoxy)-amide,6-(2,4-Dichloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide,6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazol-e-5-carboxylicacid (2-hydroxy-ethoxy)-amide,2-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide;referred to hereinafter as MEK inhibitor 2; and4-(4-bromo-2-fluorophenylamino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridazine-3-carboxamideor a pharmaceutically acceptable salt thereof. Additional illustrativeMEK/ERK inhibitors include those compounds disclosed in InternationalPublished Patent Applications WO 99/01426, WO 02/06213, WO 03/077914, WO05/051301 and WO2007/044084. In one embodiment, the MEK inhibitor isPD0325901. In another embodiment, the MEK inhibitor is U0126.

ROCK (Rho associated kinases) inhibitors refer to inhibitors of theRho-GTPase/ROCK pathway. The pathway includes the downstream proteinMyosin II, which is further downstream of ROCK (Rho-ROCK-Myosin II formsthe pathway/axis). Thus, one can use any or all of a Rho GTPaseinhibitor, a ROCK inhibitor, or a Myosin II inhibitor to achieve theeffects described herein. ROCK inhibitors suitable for use incompositions contemplated herein include, but are not limited to:thiazovivin, Y27632, fasudil, AR122-86, Y27632 H-1152, Y-30141, Wf-536,HA-1077, hydroxyl-HA-1077, GSK269962A, SB-772077-B,N-(4-Pyridyl)-N′-(2,4,6-trichlorophenyl)urea, 3-(4-Pyridyl)-1H-indole,(R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, andROCK inhibitors disclosed in U.S. Pat. No. 8,044,201, which is hereinincorporated by reference in its entirety. In one embodiment, the ROCKinhibitor is thiazovivin, Y27632, or pyrintegrin. In one embodiment, theROCK inhibitor is thiazovivin.

Activin receptor-like kinase 5 (ALK5) is the principal TGFβ receptorthat mediates cellular responses to TGFβs. Upon ligand binding,constitutively active TβRII kinase phosphorylates ALK5 which, in tum,activates the downstream signal transduction cascades. TGFβreceptor/ALK5 inhibitors can include antibodies to, dominant negativevariants of, and siRNA, microRNA, antisense nucleic acids, and otherpolynucleotides that suppress expression of, TGFβ/ALK5 receptors. TGFβreceptor/ALK5 inhibitors suitable for use in compositions contemplatedherein include, but are not limited to: SB431542; A-83-01(3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide;2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine,Wnt3a/BIO, GW788388(-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide),SM16, IN-1130(3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide),GW6604 (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine), SB-505124(2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinehydrochloride); SU5416;2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinehydrochloride (SB-505124); lerdelimumb (CAT-152); metelimumab (CAT-192);GC-1008; ID11; AP-12009; AP-11014; LY550410; LY580276; LY364947;LY2109761; SD-208; SM16; NPC-30345; Ki26894; SB-203580; SD-093; Gleevec;3,5,7,2′,4′-pentahydroxyflavone (Morin); activin-M108A; P144; solubleTBR2-Fc; and pyrimidine derivatives (see, e.g., those listed in Stiefl,et al., WO2008/006583, herein incorporated by reference). Further, while“an ALK5 inhibitor” is not intended to encompass non-specific kinaseinhibitors, an “ALK5 inhibitor” should be understood to encompassinhibitors that inhibit ALK4 and/or ALK7 in addition to ALK5, such as,for example, SB-431542 (see, e.g., Inman, et al., J, Mol. Pharmacol.62(1): 65-74 (2002). It is further believed that inhibition of theTGFβ/activin pathway will have similar effects of inhibiting ALK5. Thus,any inhibitor (e.g., upstream or downstream) of the TGFβ/activin pathwaycan be used in combination with, or instead of, ALK5 inhibitors asdescribed in each paragraph herein. Exemplary TGFβ/activin pathwayinhibitors include but are not limited to: TGFβ receptor inhibitors,inhibitors of SMAD 2/3 phosphorylation, inhibitors of the interaction ofSMAD 2/3 and SMAD 4, and activators/agonists of SMAD 6 and SMAD 7.Furthermore, the categorizations described herein are merely fororganizational purposes and one of skill in the art would know thatcompounds can affect one or more points within a pathway, and thuscompounds may function in more than one of the defined categories. Inone embodiment, the TGFβ receptor inhibitor comprises SB431542.

PDK1 or 3′-phosphoinositide-dependent kinase-1 is a master kinaseassociated with the activation of AKT/PKB and many other AGC kinasesincluding PKC, S6K, SGK. An important role for PDK1 is in the signalingpathways activated by several growth factors and hormones includinginsulin signaling. Exemplary PDK1 agonists include sphingosine (King etal, Journal of Biological Chemistry, 275: 18108-18113, 2000). Exemplaryallosteric activators of PDK1 include PS48((Z)-5-(4-Chlorophenyl)-3-phenylpent-2-enoic acid), PS08((Z)-5-(4-Bromo-2-fluorophenyl)-3-phenylpent-2-enoic acid),1-(2-(3-(4-Chlorophenyl)-3-oxo-1-phenylpropylthio)acetic acid;3,5-diphenylpent-2-enoic acids such as compound 12Z(2-(3-(4-Chlorophenyl)-3-oxo-1-phenylpropylthio)acetic acid,(Z)-5-(Napthalen-2-yl)-3-phenylpent-2-enoic acid), and compound 13Z((Z)-5-(1H-Indol-3-yl)-3-phenylpent-2-enoic acid). In one embodiment,the PDK1 agonist comprises PS48.

Mammalian target of rapamycin (mTOR) inhibitors block the activity ofthe mammalian target of rapamycin. mTOR is a protein kinase, whichregulates growth factors that stimulate cell growth and angiogenesis.mTOR inhibitors suitable for the composition and method of the presentinvention include, but not limited to rapamycin, and analogues andderivatives thereof comprising sirolimus, sirolimus derivatives,temsirolimus, 40-O-(2-hydroxy)ethyl-rapamycin (everolimus),40-O-(3-hydroxy)propyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, 40-O-tetrazole-rapamycin, andother O-alkylated or O-methylated rapamycin derivatives.

In some embodiments, the composition for improving therapeutic potentialof immune cells suitable for adoptive cell-based therapies comprises atleast one agent selected from Table 1, and derivatives and analogsthereof. In one embodiment, the composition for improving therapeuticpotential of immune cells comprises a combination of at least 2, 3, 4,5, or 6, or any number, of the agents selected from Table 1, andderivatives and analogs thereof.

In one embodiment, the composition comprising at least one agentselected from Table 1 further comprises an organic solvent. In certainembodiments, the organic solvent is substantially free of methylacetate. In certain embodiments, the organic solvent is selected fromthe group consisting of dimethyl sulfoxide (DMSO), N,N-dimethylformamide(DMF), dimethoxyethane (DME), dimethylacetamide, ethanol, andcombinations thereof. In some embodiments, the organic solvent is DMSO.In some embodiments, the organic solvent is ethanol. In some otherembodiments, the organic solvent is a mixture of DMSO and ethanol.

In some embodiments, the composition for improving therapeutic potentialof immune cells suitable for adoptive cell-based therapies comprises atleast one agent selected from Group I: dorsomorphin, heptelidic acid,1-Pyrrolidinecarbodithioic acid, and 2-DG. Without being limited to thetheory, Group I agents, among other potential roles, may impact cellmetabolism and nutrient sensing.

In some embodiments, the composition for improving therapeutic potentialof immune cells suitable for adoptive cell-based therapies comprises atleast one agent selected from Group II. GSK3 Inhibitor, ROCK inhibitor,TGFβ receptor inhibitor, MEK inhibitor, PDK1 agonist, 6-Mercaptopurine,AC-93253 iodide, tiratricol, PI-103, fulvestrant, thapsigargin, SU 4312,U0126, telmisartan, cyclosporin A,1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, BAY 61-3606,protoporphyrin IX disodium, mTOR inhibitor, TWS119, HS173, LY294002, andPictilisib. Without being limited to the theory, Group II agents, amongother potential roles, may impact signal transduction in variousfunctional pathways. In one embodiment, the agent selected from Group IIis an mTOR inhibitor. In one embodiment, the agent selected from GroupII is rapamycin, or an analogue or derivative thereof. In someembodiments, the analogues or derivatives of rapamycin include, but notlimited to, sirolimus, sirolimus derivatives, temsirolimus,40-O-(2-hydroxy)ethyl-rapamycin (everolimus),40-O-(3-hydroxy)propyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, 40-O-tetrazole-rapamycin, andother O-alkylated or O-methylated rapamycin derivatives.

In some embodiments, the composition for improving therapeutic potentialof immune cells suitable for adoptive cell-based therapies comprises atleast one agent selected from Group III: 5-Azacytidine, fludarabine,roscovitine, and PAC-1. Without being limited to the theory, Group IIIagents, among other potential roles, may impact cell proliferation andapoptosis.

In some embodiments, the composition for improving therapeutic potentialof immune cells suitable for adoptive cell-based therapies comprises atleast one agent selected from Group IV: 5,7-dichloro-8-Quinolinol,2-Naphthacenecarboxamide,7-chloro-4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahy, Nifuroxazide,and Tosufloxacin hydrochloride. Without being limited to the theory,Group IV agents, among other potential roles, may impact cell propertiesrelating to infective processes.

In some embodiments, the composition for improving therapeutic potentialof immune cells suitable for adoptive cell-based therapies comprises atleast one agent selected from Group V: sertraline,diethylenetriaminepentaacetic acid, edrophonium chloride, BIX01294,terfenadine, and dmPGE2. Without being limited to the theory, Group Vagents, among other potential roles, generally may impact other cellproperties relating to expansion, maintenance differentiation,dedifferentiation, survival rate, proliferation, cytotoxicity, cellrecall, and/or persistence.

In yet some other embodiments, the composition for improving therapeuticpotential of immune cells suitable for adoptive cell-based therapiescomprises at least one agent selected from Group I, and one or moreagents selected from Group II, Group III, Group IV, and/or Group V.

In some other embodiments, the composition for improving therapeuticpotential of immune cells suitable for adoptive cell-based therapiescomprises at least one agent selected from Group II, and one or moreagents selected from Group I, Group III, Group IV, and/or Group V. Inone embodiment, the composition comprise an agent selected from Group IIand an agent selected from Group V. In one embodiment, the agentselected from Group II is an mTOR inhibitor. In one embodiment, theagent selected from Group II is rapamycin, or an analogue or derivativethereof. In some embodiments, the analogues or derivatives of rapamycininclude, but not limited to, sirolimus, sirolimus derivatives,temsirolimus, 40-O-(2-hydroxy)ethyl-rapamycin (everolimus),40-O-(3-hydroxy)propyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, 40-O-tetrazole-rapamycin, andother O-alkylated or O-methylated rapamycin derivatives. In oneembodiment the agent selected from Group V is dmPGE2, or an analogue orderivative thereof.

In yet some other embodiments, the composition for improving therapeuticpotential of immune cells suitable for adoptive cell-based therapiescomprises at least one agent selected from Group III, and one or moreagents selected from Group I, Group II, Group IV, and/or Group V.

In still some other embodiments, the composition for improvingtherapeutic potential of immune cells suitable for adoptive cell-basedtherapies comprises at least one agent selected from Group IV, and oneor more agents selected from Group I, Group II, Group III, and/or GroupV.

In still some other embodiments, the composition for improvingtherapeutic potential of immune cells suitable for adoptive cell-basedtherapies comprises at least one agent selected from Group IV, and oneor more agents selected from Group I, Group II, Group III, and/or GroupIV.

In some embodiments, the composition for improving therapeutic potentialof immune cells suitable for adoptive cell-based therapies comprises atleast one agent selected from a group consisting of a GSK3 inhibitor, aMEK inhibitor, a ROCK inhibitor, a TGFβ inhibitor, a PDK1 agonist, andan mTOR inhibitor.

In some embodiments, the composition comprises a combination of two ormore agents selected from Table 1, wherein the agents have additiveeffect in the combination. As defined, “additive” refers to when two ormore agents in a combination produce an effect nearly equal to the sumof their individual effects. In some embodiments, one or more of theagents in a combination are from the same group: Group I, II, III, IV,or V. In some embodiments, one or more of the agents in a combinationare from different groups.

In some embodiments, the composition for improving therapeutic potentialof immune cells comprises a combination of rapamycin and dmPGE2, or anycombination of their respective analogues and derivatives.

In some embodiments, the composition comprises a synergistic combinationof two or more agents selected from Table 1. As defined, “synergy” is anenhanced effect such that the working together of two or more agents toproduce an effect greater than the sum of their individual effects. Inone embodiment, the composition comprising a synergistic combinationcomprises at least one agent selected from the group consisting ofTWS119, HS173, LY294002, Pictilisib, and 2-DG. In one embodiment, thecomposition comprises a combination comprising at least one agentselected from the group consisting of TWS119, HS173, LY294002,Pictilisib, and 2-DG, and one or more additional agent selected from thegroup of compounds listed in Table 1. In one embodiment, the compositioncomprising TWS119, further comprises two or more additional agentsselected from Table 1. In one embodiment, the composition comprisingHS173, further comprises two or more additional agents selected fromTable 1. In one embodiment, the composition comprising LY294002, furthercomprises two or more additional agents selected from Table 1. In oneembodiment, the composition comprising Pictilisib, further comprises twoor more additional agents selected from Table 1. In one embodiment, thecomposition comprising 2-DG, further comprises two or more additionalagents selected from Table 1.

In some embodiments, the composition comprising one or more agentsselected from the group consisting of the compounds listed in Table 1,further comprises one of more additional additives selected from thegroup consisting of peptides, cytokines, mitogens, growth factors, smallRNAs, dsRNAs (double stranded RNA), mononuclear blood cells, feedercells, feeder cell components or replacement factors, vectors comprisingone or more polynucleic acids of interest, antibodies and antibodyfragments thereof, and/or chemotherapeutic agent or radioactive moiety.In some embodiments, the additional additive comprises an antibody, oran antibody fragment. In some of these embodiments, the antibody, orantibody fragment, specifically binds to a viral antigen. In otherembodiments, the antibody, or antibody fragment, specifically binds to atumor antigen.

In some embodiments, the cytokine and growth factor comprise one or moreof the following cytokines or growth factors: epidermal growth factor(EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growthfactor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growthfactor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growthfactor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor(NGF), platelet-derived growth factor (PDGF), transforming growth factorbeta (TGF-β), vascular endothelial cell growth factor (VEGF)transferrin, various interleukins (such as IL-1 through IL-18), variouscolony-stimulating factors (such as granulocyte/macrophagecolony-stimulating factor (GM-CSF)), various interferons (such asIFN-7), stem cell factor (SCF) and erythropoietin (Epo). In someembodiments, the cytokine comprises at least interleukin-2 (IL-2),interleukin 7 (IL-7), interleukin-12 (IL-12), interleukin-15,interleukin 18 (IL-18), interleukin 21 (IL-21), or any combinationsthereof. In some embodiments, the growth factor of the compositioncomprises fibroblast growth factor. These cytokines may be obtainedcommercially, for example from R&D Systems (Minneapolis, Minn.), and maybe either natural or recombinant. In particular embodiments, growthfactors and cytokines may be added at concentrations contemplatedherein. In certain embodiments growth factors and cytokines may be addedat concentrations that are determined empirically or as guided by theestablished cytokine art.

In some embodiments, the mitogen of the composition comprisesconcanavalin A. In some other embodiments, the feeder cells aregenetically modified. In some embodiments, the feeder cells comprise oneor more of the followings: mononuclear blood cells, thymic epithelialcells, endothelial cells, fibroblasts, leukemic cells K562, Raji cells,or feeder cell components or replacement factors thereof.

In some embodiments, the small RNA comprises one or more of siRNA,shRNA, miRNA and antisense nucleic acids. In some other embodiments, thesmall RNA comprises one or more of the followings: miR-362-5p,miR-483-3p, miR-210 and miR-598.

In some embodiments, the vector comprising one or more polynucleic acidsof interest is integrating or non-integrating. In some embodiments, thevector comprising one or more polynucleic acids of interest furthercomprises backbones of an adenovirus vector, plasmid vector,adeno-associated virus vector, retrovirus vector, lentivirus vector,Sendai virus vector, episomal vector and the like. In some embodiments,the plasmid vectors for the expression in animal cells include, forexample, pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo, and the like. Insome embodiments, the one or more polynucleic acids comprised in thevector encode one or more proteins or polypeptides. In some embodiments,the one or more polynucleic acids encode Delta-like 1 (DLL1), Delta-like3 (DLL3), Delta-like 4 (DLL4), Jagged1 (Jag1), or Jagged2. In someembodiments, the one or more polynucleic acids encode Jagged 1.

II. Immune Cells for Adoptive Cellular Therapies

The present invention provides a composition comprising an isolatedpopulation or subpopulation of immune cells that have been contactedwith one or more agents selected from Table 1. In one embodiment, theisolated population or subpopulation of immune cells have been contactedwith one or more agents selected from Table 1 in an amount sufficient toimprove the therapeutic potential of the immune cells. In someembodiments, the treated immune cells are used in a cell-based adoptivetherapy. The present invention further provides a population orsubpopulation of immune cells, and one or more agents selected from theagents listed in Table 1, wherein a treatment of the population orsubpopulation of immune cells using the one or more agents selected fromthe agents listed in Table 1 improves the therapeutic potential of theimmune cells for adoptive therapy. The treatment can modify thebiological properties of the immune cells to improve cell proliferation,cytotoxicity, and persistence, and/or reduce the relapse rate of thecell therapy.

In some embodiments, the population of immune cells comprises T cells.In some embodiments, the population of immune cells comprises NK cells.In some embodiments, the population of immune cell comprises NKT cell.

In some embodiments, a population or subpopulation of T cells contactedwith one or more agents selected from Table 1 comprises an increasednumber or ratio of naïve T cells (Tn), stem cell memory T cells (Tscm),and/or central memory T cells (Tcm), and/or improved cell proliferation,cytotoxicity, cell recall, and/or persistence in comparison to the Tcells without the same treatment. In some embodiments the number of Tn,Tscm, and/or Tcm is increased by at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or increased by at least 5, 10, 15, or 20 fold comparedto the number of Tn, Tscm, and/or Tcm in the cell population without thesame treatment with one or more agents selected from Table 1.

In some embodiments, a population or subpopulation of NK cells contactedwith one or more agents selected from Table 1 comprises an increasednumber or ratio of adaptive (or memory) NK cells, and/or improved cellproliferation, cytotoxicity, cell recall, and/or persistence incomparison to the NK cells without the same treatment. In someembodiments the number of adaptive NK cells is increased by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or increased by at least 5,10, 15, or 20 fold compared to the number of adaptive NK cells in thecell population without the same treatment with one or more agentsselected from Table 1. In one embodiment, a population or subpopulationof NK cells contacted with a GSK3 inhibitor, a MEK inhibitor, a ROCKinhibitor, a TGFβ inhibitor, a PDK1 agonist, and/or rapamycin comprisesan increased number or ratio of adaptive NK cells. In one embodiment,the adaptive NK cell is characterized by CD3- and CD56+, and at leastone of CD57+, NKG2C+, low PLZF, low SYK, low FεFRγ, low EAT-2, lowTIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and lowCD45RA. In some embodiments, the adaptive NK cells are at least two ofCD57+, NKG2C+, low PLZF, low SYK, low FεFRγ, low EAT-2, low TIGIT, lowPD1, low CD7, low CD161, high LILRB1, high CD45RO, and low CD45RA. Forexample, the adaptive NK cell can be CD57+ and NKG2C+. In someembodiments, the adaptive NK cells are at least three of CD57+, NKG2C+,low PLZF, low SYK, low FεFRγ, low EAT-2, low TIGIT, low PD1, low CD7,low CD161, high LILRB1, high CD45RO, and low CD45RA. For example, theadaptive NK cell can be SYK-, FεFRγ-, and EAT-2-. In one embodiment, theGSK-3β inhibitor is CHIR99021, BIO, TWS119, or Kenpaullone. In oneembodiment, the GSK-3β inhibitor is TWS119. In another embodiment, theGSK-3β inhibitor is CHIR99021. In yet another embodiment the GSK-3βinhibitor is BIO. In one embodiment, the mTOR inhibitor is rapamycin, oran analogue or a derivative thereof comprising sirolimus, sirolimusderivatives, temsirolimus, 40-O-(2-hydroxy)ethyl-rapamycin (everolimus),40-O-(3-hydroxy)propyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, 40-O-tetrazole-rapamycin, andother O-alkylated or O-methylated rapamycin derivatives.

In some other embodiments, a population or subpopulation of NKT cellscontacted with one or more agents selected from Table 1 comprises anincreased number or ratio of type I NKT cells vs type II, and/orimproved cell proliferation, cytotoxicity, cell recall, and/orpersistence in comparison to the isolated population or subpopulation ofNKT cells without the treatment with one or more agents selected fromTable 1. In some embodiments the number of type I NKT cells is increasedby at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or increased byat least 5, 10, 15, or 20 fold compared to the number of type I NKTcells in the cell population without the same treatment with one or moreagents selected from Table 1.

In some embodiments, the increased number or ratio of naïve T cells(Tn), stem cell memory T cells (Tscm), central memory T cells (Tcm),adaptive NK cells, and/or type I NKT cells are due to improvedmaintenance and expansion of these cell subtypes, and/or increased celldedifferentiation/reprogramming from more mature cell subtypes to cellsubtypes in a desired differentiation state.

In some embodiments, after contacting a population of immune cell withone or more of the agents included in Table 1, the number of naïve Tcells (Tn), stem cell memory T cells (Tscm), central memory T cells(Tcm) in the population is increased in comparison to untreated immunecell population, wherein the Tn, Tscm and Tcm are characterized byco-expression of CCR7 and/or CD62L.

In some embodiments, after contacting a population of immune cells withone or more of the agents included in Table 1, the number of adaptive NKcells in the population is increased in comparison to untreated immunecell population, wherein the adaptive NK cells are characterized byCD3−, CD56+, CD16+, NKG2C+, and CD57+. In some other embodiments, theadaptive NK cells are characterized by CD3−, CD56+, and at least one,two or three of CD57+, NKG2C+, low PLZF, low SYK, low FεFRγ, low EAT-2,low TIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, andlow CD45RA.

In some embodiments, after contacting a population of immune cells withone or more of the agents included in Table 1, the number of type I NKTcells in the population is increased in comparison to untreated immunecell population, wherein the type I NKT cells are characterized bysurface antigens CD3+, CD56+, TCR Vα24+, and/or TCR Vβ11+.

In some embodiments, the population or subpopulation of T, NK or NKTcells for treatment by the agents disclosed herein can be isolated froma human or a non-human mammal. Examples of such non-human mammalsinclude, but are not limited to rabbit, horse, bovine, sheep, pigs,dogs, cats, mice, rats, and transgenic species thereof.

The population or subpopulation of T cells can be obtained or isolatedfrom a number of sources, including but not limited to peripheral blood,bone marrow, lymph node tissue, cord blood, thymus tissue, and tissuefrom a site of infection, ascites, pleural effusion, spleen tissue, andtumors. The bone marrow can be obtained from femurs, iliac crest, hip,ribs, sternum, and other bones. In addition, the T cell lines availablein the art can also be used, such as Jurkat, SupT1, and others.

The population or subpopulation of NK cells can be obtained, or can beenriched, from a number of sources, including but not limited toperipheral blood, cord blood, and tumors.

Fully mature NKT cells can be obtained, or can be enriched, fromperipheral blood, with smaller populations of mature NKT cellspotentially found in bone marrow, lymph node tissue and cord blood,thymus tissue.

In certain embodiments of the present invention, an isolated or enrichedpopulation or subpopulation of T, NK, NKT cells can be obtained from aunit of blood using any number of techniques known to the skilledartisan, such as Ficoll™ separation. In one embodiment, T, NK or NKTcells from the circulating blood of an individual are obtained byapheresis. The apheresis product typically contains cells, including Tcells, monocytes, granulocytes, B cells, NK cells, NKT cells, othernucleated white blood cells, red blood cells, and platelets. In oneembodiment, the cells collected by apheresis can be washed to remove theplasma fraction and to place the cells in an appropriate buffer or mediafor subsequent processing steps. In one embodiment of the invention, thecells are washed with phosphate buffered saline (PBS). In an alternativeembodiment, the wash solution lacks calcium and can lack magnesium orcan lack many if not all divalent cations. As those of ordinary skill inthe art would readily appreciate a washing step can be accomplished bymethods known to those in the art, such as by using a semi-automated“flow-through” centrifuge (for example, the Cobe 2991 cell processor,the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to themanufacturer's instructions. After washing, the cells can be resuspendedin a variety of biocompatible buffers, such as, for example, Ca-free,Mg-free PBS, PlasmaLyte A, or other saline solution with or withoutbuffer. Alternatively, the undesirable components of the apheresissample can be removed and the cells directly resuspended in culturemedia.

In another embodiment, the population or subpopulation of T, NK or NKTcells are isolated or enriched from peripheral blood lymphocytes bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient or by counterflow centrifugalelutriation.

In one embodiment, a specific subpopulation of T cells, can be furtherisolated or enriched by positive or negative selection techniques suchas CD3, CD28, CD4, CD8, CD45RA, CD45RO, CD62L, CCR7, CD27, and/or CD122antibodies. For example, in one embodiment, the isolated or enrichedpopulation or subpopulation of T cells are expanded and activated byincubation with anti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, suchas DYNABEADS® M-450 CD3/CD28 T, for a time period sufficient forpositive selection of the desired T cells. In one embodiment, the timeperiod is about 30 minutes. In a further embodiment, the time periodranges from 30 minutes to 72 hours or longer and all integer valuesbetween. In a further embodiment, the time period is at least 1, 2, 3,4, 5, or 6 hours. In yet another preferred embodiment, the time periodis 10 to 72 hours. In one preferred embodiment, the incubation timeperiod is 24 hours. For isolation of T cells from patients withleukemia, use of longer incubation times, such as 24 hours, can increasecell yield. Longer incubation times can be used to isolate T cells inany situation where there are few T cells as compared to other celltypes, such in isolating tumor infiltrating lymphocytes (TIL) from tumortissue or from immunocompromised individuals. Further, use of longerincubation times can increase the efficiency of capture of CD8+ T cells.Thus, by simply shortening or lengthening the time T cells are allowedto bind to the CD3/CD28 beads and/or by increasing or decreasing theratio of beads to T cells (as described further herein), specificpopulations or subpopulations of T cells can be further selected for oragainst at culture initiation or at other time points during theprocess. Additionally, by increasing or decreasing the ratio of anti-CD3and/or anti-CD28 antibodies on the beads or other surface, specificpopulations or subpopulations of T cells can be preferentially selectedfor or against at culture initiation or at other desired time points.The skilled artisan would recognize that multiple rounds of selectioncan also be used in the context of this invention. In certainembodiments, it can be desirable to perform the selection procedure anduse the “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Isolation or enrichment of a population or subpopulation of T, NK or NKTcells by negative selection can be accomplished with a combination ofantibodies directed to surface markers unique to the negatively selectedcells. One method is cell sorting and/or selection via negative magneticimmuno-adherence or fluorescence-activated cell sorting that uses acocktail of monoclonal antibodies directed to cell surface markerspresent on the cells negatively selected. For example, to enrich forCD3+ cells by negative selection, a monoclonal antibody cocktailtypically includes antibodies to CD14, CD20, CD11b, CD16, and HLA-DR. Incertain embodiments, it can be desirable to enrich for or positivelyselect for regulatory T cells which typically express CD4+, CD25+,CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain embodiments, Tregulatory cells are depleted by anti-CD25 conjugated beads or othersimilar method of selection. In some embodiments, a desired T cellsubpopulation for immunotherapy is enriched or selected from themodulated immune cells comprising T cells by CCR7 and CD62L.Alternatively cells of interest may be selected according to physicalparameters including differential size, density, granularity,deformability, resistance or capacitance.

In one embodiment, a population or subpopulation of adaptive NK cellsare enriched by selecting within the modulated immune cells comprisingNK cells for those phenotypically CD3- and CD56+, using the identifierssuch as include positive expression of CD16, NKG2C, and CD57. Further,negative selection of adaptive subpopulation can be based on lack ofexpression of NKG2C and/or CD57, and additionally lack expression of oneor more of the following: low PLZF, low SYK, low FcεRγ, low EAT-2, lowTIGIT, low PD1, low CD7, low CD161, high LILRB1, high CD45RO, and lowCD45RA.

In one embodiment, a population or subpopulation of NKT cells areenriched by selecting within the population of NK cells for thosephenotypically expressing the invariant TCRα chain, and specifically thefollowing combination of markers: CD3+, CD56+, TCR Vα24+, and/or TCRVo11+. Alternatively, NKT cells can be selected based on a combinationof phenotype combined with expression of the invariant TCRα chain.

The blood samples or apheresis product from a subject can be collectedat a time period prior to when the immune cells as described herein areisolated. As such, the source of the cells to be modulated can becollected at any time point necessary, and desired cells, such as Tcells, NK cells and NKT cells, isolated and frozen for later use incell-based immunotherapy for any number of diseases or conditions thatwould benefit from such cell therapy, such as those described herein. Inone embodiment a blood sample or an apheresis product is collected froma generally healthy subject. In certain embodiments, a blood or anapheresis product is collected from a generally healthy subject who isat risk of developing a disease, but who has not yet developed adisease, and the cells of interest are isolated and frozen for lateruse. In other embodiments, a blood sample or an apheresis product iscollected from a subject who has been previously administered withgenetically modified immune cells (genetically engineered or naturallyderived from rearrangements, mutations, genetic imprinting and/orepigenetic modification). In certain embodiments, the T, NK, NKT orother immune cells can be expanded, frozen, and treated and used at alater time. In certain embodiments, samples are collected from a patientshortly after diagnosis of a particular disease as described herein butprior to any treatments. In some embodiments, the cells are isolatedfrom a subject presenting CMV (cytomegalovirus) seropositivity. In afurther embodiment, the cells are isolated from a blood or an apheresisproduct from a subject prior to any number of relevant treatmentmodalities, including but not limited to treatment with agents such asnatalizumab, efalizumab, antiviral agents, chemotherapy, radiation,immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan,fludarabine, cyclosporin, FK506, mycophenolic acid, steroids, FR901228,and irradiation. In a further embodiment, the cells are isolated from apatient and frozen for later use in conjunction with (e.g., before,simultaneously or following) bone marrow or stem cell transplantation, Tcell ablative therapy using either chemotherapy agents such as,fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, orantibodies such as OKT3 or CAMPATH. In another embodiment, the cells areisolated prior to and can be frozen for later use for treatmentfollowing B-cell ablative therapy such as agents that react with CD20,e.g., Rituxan.

In some embodiments, the population or subpopulation of T, NK or NKTcells are genomically engineered, which include insertion, deletion, ornucleic acid replacement. Modified immune cells may express cytokinetransgenes, silenced inhibitory receptors; or overexpress activatingreceptors, or CARs for retargeting the immune cells. In someembodiments, the population of immune cells isolated for modulation froma subject, or donor, or isolated from or comprised in peripheral blood,bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from asite of infection, ascites, pleural effusion, spleen tissue, tumors of asubject/donor may be genetically modified. In some embodiments, theisolated population of immune cells are genomically engineered andcomprise an insertion, a deletion, and/or a nucleic acid replacement. Insome particular embodiments, the immune cells comprise an exogenousnucleic acid encoding a T Cell Receptor (TCR), a Chimeric AntigenReceptor (CAR), and/or overexpression of CD16 or a variant thereof.

The genomically engineered immune cells comprise genetically modifiedmodalities including one or more of: safety switch proteins, targetingmodalities, receptors, signaling molecules, transcription factors,pharmaceutically active proteins and peptides, drug target candidates;or proteins promoting engraftment, trafficking, homing, viability,self-renewal, persistence, immune response regulation and modulation,and/or survival of the immune cells. In some other embodiments, thegenetically modified modalities include one or more of (i) deletion orreduced expression of B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3,RFXANK, CIITA, RFX5, or RFXAP, and any gene in the chromosome 6p21region; (ii) introduced or increased expression of HLA-E, HLA-G, HACD16,hnCD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1,A2AR, Fc receptor, or surface triggering receptors for coupling with bi-or multi-specific or universal engagers. In some embodiments, the T, NKor NKT cells comprise an exogenous nucleic acid. In some embodiments,the exogenous nucleic acid is introduced to the immune cells via directgenomic editing of the cells. In some other embodiments, the exogenousnucleic acid is introduced to the immune cells via retaining the samefrom a genomically engineered hematopoietic stem or progenitor cell oriPSC, which gives rise to the immune cell through differentiation. Insome embodiments, the exogenous nucleic acid for a T cell can encode aTCR (T Cell Receptor), a CAR (Chimeric Antigen Receptor), a bi-specificT cell engager (BiTE), a tri-specific T cell engager, a multi-specific Tcell engager, or a universal engager compatible with multiple immunecell types. In some embodiments, the exogenous nucleic acid for a NKcell can encode a TCR, a CAR, a CD16 or a variant thereof, a NY-ESO, abi-specific killer cell engager (BiKE), a tri-specific killer cellengager (TriKE), a multi-specific killer cell engager, or a universalengager compatible with multiple immune cell types. In some embodiments,the exogenous nucleic acid for a NKT cell can be an altered TCR or CAR.In some embodiments, the exogenous nucleic acid encoding CAR19. In someembodiments, CD16 variants comprise high-affinity CD16 (HACD16),non-cleavable CD16, and high-affinity non-cleavable CD16 (hnCD16).

In some embodiments, the population or subpopulation of immune cells formodulation is differentiated in vitro from a stem cell or progenitorcell. In some embodiments, the isolated population or subpopulation ofT, NK or NKT cells can be differentiated from a stem cell, ahematopoietic stem or progenitor cell (HSC), or a progenitor cell. Theprogenitor cell can be a CD34+ hemogenic endothelium cell, a multipotentprogenitor cell, a T cell progenitor, a NK cell progenitor, or a NKTcell progenitor. The stem cell can be a pluripotent stem cell, such asinduced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs).The iPSC is a non-naturally occurring reprogrammed pluripotent cell.Once the cells of a subject have been reprogrammed to a pluripotentstate, the cells can then be programmed or differentiated to a desiredcell type or subtypes, such as T, NK, or NKT cells.

In some embodiments, the iPSC is differentiated to a T, NK or NKT cellsby a multi-stage differentiation platform wherein cells from variousstages of development can be induced to assume a hematopoieticphenotype, ranging from mesodermal stem cells, to fully differentiatedT, NK or NKT cells (See e.g. U.S. Applications 62/107,517 and62/251,016, the disclosures of which are incorporated herein in theirentireties). In some embodiments, the iPSC, HSC, or progenitor for T, NKor NKT cell differentiation is genomically engineered, which includeinsertion, deletion, or nucleic acid replacement.

In some embodiments, the genomically engineered iPSC, HSC orhematopoietic progenitor cells comprise genetically modified modalitiesincluding one or more of: safety switch proteins, targeting modalities,receptors, signaling molecules, transcription factors, pharmaceuticallyactive proteins and peptides, drug target candidates; or proteinspromoting engraftment, trafficking, homing, viability, self-renewal,persistence, immune response regulation and modulation, and/or survivalof the iPSC, HSC, progenitor, or their derived cells. In some otherembodiments, the genetically modified modalities include one or more of(i) deletion or reduced expression of B2M, TAP1, TAP2, Tapasin, NLRC5,PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, and any gene in thechromosome 6p21 region; (ii) introduced or increased expression ofHLA-E, HLA-G, HACD16, hnCD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131,CD137, CD80, PDL1, A2AR, Fc receptor, surface triggering receptors forcoupling with bi- or multi-specific or universal engagers, a TCR (T CellReceptor), or a CAR (Chimeric Antigen Receptor). In some embodiments,the iPSC, HSC, or progenitor for T, NK or NKT cell differentiationcomprises modified HLA class I and/or II. In some embodiments, the iPSC,HSC, or progenitor for T, NK or NKT cell differentiation with modifiedHLA class I and/or II comprises null or low expression of at least oneof B2M, HLA-E/G, PDL1, A2AR, CD47, LAG3, TIM3, TAP1, TAP2, Tapasin,NLRC5, PD1, RFKANK, CIITA, RFX5, and RFXAP. In some embodiments, theiPSC, HSC, or progenitor for T, NK or NKT cell differentiation has anexogenous nucleic acid. In some embodiments, the exogenous nucleic acidcan encode, a bi-specific T cell engager (BiTE), a tri-specific T cellengager, a multi-specific T cell engager, a CD16 or a variant thereof, aNY-ESO, a bi-specific killer cell engager (BiKE), a tri-specific killercell engager (TriKE), a multi-specific killer cell engager, or auniversal engager compatible with multiple immune cell types. In someembodiments, the exogenous nucleic acid encoding hnCD16 in the iPSC,HSC, or progenitor for T, NK or NKT cell differentiation. In someembodiments, the exogenous nucleic acid encoding CAR19 in the iPSC, HSC,or progenitor for T, NK or NKT cell differentiation.

In some embodiments, the population or subpopulation of immune cells istrans-differentiated in vitro from a non-pluripotent cell ofnon-hematopoietic fate to a hematopoietic lineage cell or from anon-pluripotent cell of a first hematopoietic cell type to a differenthematopoietic cell type, which can be a T, NK, or NKT progenitor cell ora fully differentiated specific type of immune cell, such as T, NK, orNKT cell (See e.g. U.S. Pat. No. 9,376,664 and U.S. application Ser. No.15/072,769, the disclosure of which is incorporated herein in theirentirety). In some embodiments, the non-pluripotent cell ofnon-hematopoietic fate is somatic cells, such as skin fibroblasts,adipose tissue-derived cells and human umbilical vein endothelial cells(HUVEC). Somatic cells useful for trans-differentiation may beimmortalized somatic cells.

Various strategies are being pursued to induce pluripotency, or increasepotency, in cells (Takahashi, K., and Yamanaka, S., Cell 126, 663-676(2006); Takahashi et al., Cell 131, 861-872 (2007); Yu et al., Science318, 1917-1920 (2007); Zhou et al., Cell Stem Cell 4, 381-384 (2009);Kim et al., Cell Stem Cell 4, 472-476 (2009); Yamanaka et al., 2009;Saha, K., Jaenisch, R., Cell Stem Cell 5, 584-595 (2009)), and improvethe efficiency of reprogramming (Shi et al., Cell Stem Cell 2, 525-528(2008a); Shi et al., Cell Stem Cell 3, 568-574 (2008b); Huangfu et al.,Nat Biotechnol 26, 795-797 (2008a); Huangfu et al., Nat Biotechnol 26,1269-1275 (2008b); Silva et al., Plos Bio 6, e253. Doi: 10.1371/journal.Pbio. 0060253 (2008); Lyssiotis et al., PNAS 106, 8912-8917 (2009);Ichida et al., Cell Stem Cell 5, 491-503 (2009); Maherali, N.,Hochedlinger, K., Curr Biol 19, 1718-1723 (2009b); Esteban et al., CellStem Cell 6, 71-79 (2010); and Feng et al., Cell Stem Cell 4, 301-312(2009)), the disclosures of which are hereby incorporated by referencein their entireties.

III. Method of Modulating Immune Cells for Adoptive Therapies

The present invention provides a method of modulating a population or asubpopulation of immune cells suitable for adoptive cell-basedtherapies, and the method comprises contacting the immune cells with acomposition comprising at least one agent selected from Table 1.

In one embodiment, the method of modulating a population or asubpopulation of immune cells suitable for adoptive cell-based therapiescomprises contacting the immune cells with a composition comprising atleast one agent selected from Table 1 wherein the contacted immune cellshave increased cell expansion, increased number or ratio of one or moredesired cell subpopulations, and/or improved proliferation,cytotoxicity, cell recall, and/or persistence in comparison to immunecells without contacting the agents of Table 1.

In some embodiments, the method of modulating a population or asubpopulation of immune cells suitable for adoptive cell-based therapiescomprises contacting the immune cells with a composition comprising atleast one agent selected from Table 1, wherein the maintenance andexpansion of one or more desired subpopulation of cells are improved incomparison to immune cells without contacting the agents of Table 1.

In some embodiments, the method of modulating a population or asubpopulation of immune cells suitable for adoptive cell-based therapiescomprises contacting the immune cells with a composition comprising atleast one agent selected from Table 1, wherein the number or ratio ofimmune cells in the population reprogrammed to a desired state ofdifferentiation is increased in comparison to immune cells withoutcontacting the agents of Table 1.

In some embodiments, the method of modulating a population or asubpopulation of immune cells suitable for adoptive cell-based therapiescomprises contacting the immune cells with a composition comprising atleast one agent selected from Table 1 in a sufficient amount forincreasing cell expansion, increasing number or ratio of one or moredesired immune cell subpopulations, and/or improving proliferation,cytotoxicity, cell recall, and/or persistence of the immune cell incomparison to immune cells without contacting the agents of Table 1. Inone embodiment, the agent for immune cell treatment is between about 0.1nM to about 50 μM. In one embodiment, the agent for immune celltreatment is about 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500nM, 1 μM, 5 μM, 10 μM, 20 μM, or 25 μM, or any concentration in-between.In one embodiment, the agent for immune cell treatment is between about0.1 nM to about 5 nM, is between about 1 nM to about 100 nM, is betweenabout 50 nM to about 250 nM, between about 100 nM to about 500 nM,between about 250 nM to about 1 μM, between about 500 nM to about 5 μM,between about 3 μM to about 10 μM, between about 5 μM to about 15 μM,between about 12 μM to about 20 μM, or between about 18 μM to about 25μM.

In some embodiments, the method of modulating a population or asubpopulation of immune cells suitable for adoptive cell-based therapiescomprises contacting the immune cells with a composition comprising atleast one agent selected from Table 1 for a sufficient length of timefor increasing cell expansion, increasing number or ratio of one or moredesired immune cell subpopulations, and/or improving proliferation,cytotoxicity, cell recall, and/or persistence of the immune cell incomparison to immune cells without contacting the agents of Table 1. Inone embodiment, the immune cells are contacted with one or more agent ofTable 1 for at least 10 minutes, 30 minutes, 1 hours, 2, hours, 5 hours,12 hours, 16 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 10 days, 15 days, 20 days, 25 days, 30 days, or any lengthof period in between. In one embodiment, the immune cells are contactedwith one or more agent of Table 1 for between about 0.5 hour to about 2hours, between about 1 hour to about 12 hours, between about 10 hours toabout 2 days, between about 1 day to about 3 days, between about 2 daysto about 5 days, between about 3 days to about 6 days, between about 5days to about 8 days, between about 7 days to about 14 days, betweenabout 12 days to about 22 days, between about 14 days to about 25 days,between about 20 days to about 30 days. In some embodiments, the immunecells are contacted with one or more agent of Table 1 for no less than16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2hours, or any length of time in between. As such, said sufficient lengthof time, for example, is no less than 15, 13, 11, 9, 7, 5, 3, or 1hour(s). In some other embodiments of the method, said sufficient lengthof time is no less than 24 hours, 36 hours, 48 hours, 60 hours, 72hours, or any length of time in between. As such, said sufficient lengthof time is, for example, no less than 30, 42, 54, 66, 78, 90 hour(s).

The method of modulating a population or a subpopulation of immune cellssuitable for adoptive cell-based therapies that comprises contacting theimmune cells with a composition comprising at least one agent selectedfrom Table 1, may further comprise enriching or isolating one or moredesired subpopulations from the immune cells after the contacting,wherein the one or more desired subpopulations are selected from thegroup consisting of naïve T cell, stem cell memory T cell, centralmemory T cell, adaptive NK cell, and type I NKT cell.

In some embodiments, the subject is CMV seropositive, or may have beenpreviously administered with genetically modified immune cells. In someembodiments, the subject may be CMV seropositive. In some otherembodiments, the isolated immune cells for modulation are geneticallymodified (genetically engineered or naturally derived fromrearrangements, mutations, genetic imprinting and/or epigeneticmodification). In some embodiments, the isolated immune cells formodulation comprise at least one genetically modified modality. In someembodiments, the isolated population of immune cells are genomicallyengineered and comprise an insertion, a deletion, and/or a nucleic acidreplacement. In some particular embodiments, the immune cells comprisean exogenous nucleic acid encoding a T Cell Receptor (TCR), a ChimericAntigen Receptor (CAR), and/or overexpression of CD16 or a variantthereof. As such, the genetically modified immune cells are isolated forex vivo modulation using the present compositions and methods asdisclosed. In some embodiments, after modulation, the geneticallymodified immune cells isolated from a subject may be administered to thesame donor or a different patient. In some embodiments, the donorderived immune cells for modulation comprise an exogenous nucleic acidencoding a T Cell Receptor (TCR) and/or a Chimeric Antigen Receptor(CAR).

Alternatively, the population of immune cells for modulation may bedifferentiated in vitro from stem cell, hematopoietic stem or progenitorcells, or progenitor cells; or trans-differentiated from anon-pluripotent cell of hematopoietic or non-hematopoietic lineage. Insome embodiments, the stem cells, hematopoietic stem or progenitorcells, progenitor cells, or a non-pluripotent cell that derive theimmune cells for modulation are genomic engineered and comprise aninsertion, a deletion, and/or a nucleic acid replacement, as such thederived immune cells for modulation comprise the same genetic modalitiesintroduced by genomic engineering in the source cells.

IV. Therapeutic Use of the Treated Immune Cells, Immune Cell Populationor Subpopulations

The present invention provides a composition comprising an isolatedpopulation or subpopulation of immune cells that have been contactedwith one or more agents selected from Table 1 in an amount sufficient toimprove the therapeutic potential of the immune cells when used in acell based adoptive therapy. In one embodiment, the isolated populationor subpopulation of immune cell that has been treated comprises anincreased number or ratio of naïve T cells, stem cell memory T cells,and/or central memory T cells. In one embodiment, the isolatedpopulation or subpopulation of immune cell that has been contactedcomprises an increased number or ratio of type I NKT cells. In anotherembodiment, the isolated population or subpopulation of immune cell thathas been contacted comprises an increased number or ratio of adaptive NKcells. It is contemplated herein that combination treatments using NKcell therapy products together with other drugs that target tumor cellsor modulate cytotoxic activity of NK cells. In some embodiments of thecomposition, the composition further comprises one of more additionaladditives selected from the group consisting of peptides, cytokines,mitogens, growth factors, small RNAs, dsRNAs (double stranded RNAs),mononuclear blood cells, feeder cells, feeder cell components orreplacement factors, vectors comprising one or more polynucleic acids ofinterest, antibodies, chemotherapeutic agents or radioactive moiety, andimmunomodulatory drugs (IMiDs).

The present invention also provides compositions and methods ofcombinational treatment comprising the immune cells modulated with oneor more agents comprising the compounds listed in Table 1, andadditional therapeutic agents. In some embodiments, the additionaltherapeutic agent comprises an antibody, or an antibody fragment. Insome embodiments, the antibody may be a humanized antibody, a humanizedmonoclonal antibody, a chimeric antibody. In some embodiments, theantibody, or antibody fragment, specifically binds to a viral antigen.In other embodiments, the antibody, or antibody fragment, specificallybinds to a tumor antigen. In some embodiments, the tumor or viralspecific antigen activates the modulated NK cells to make use ofantibody-dependent cellular cytotoxicity (ADCC) and lysis of the targetcell. Monoclonal antibodies (mAbs) bind to the target cell plus engagingCD16 on NK cells and other cell types resulting in killing of tumor cellby ADCC both in vivo and in vitro. mAbs can also enhance ADCC andstimulate NK cells by blocking NK cell inhibition. In some embodiments,the NK cell mediated ADCC is through expressed CD16 and geneticallyengineered variants thereof by the modulated NK cells. The geneticallyengineered variants of CD16 include, but are not limited to,non-cleavable CD16, high affinity CD16 (haCD16), and high affinitynon-cleavable CD16 (hnCD16). As such, the above aspect of the presentinvention provides GSK3i modulated NK cells capable of performing ADCCin antibody combination cancer treatments. In some embodiments, theantibodies suitable for combinational treatment with anti-cancer NKcells provided herein include, but are not limited to, anti-CD20(retuximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab,obinutuzumab), anti-Her2 (trastuzumab), anti-CD52 (alemtuzumab),anti-EGFR (certuximab), and anti-CD38 (daratumumab), and their humanizedand Fc modified variants. Additionally, the design of bi- andtrispecific antibodies, fusing the Fab region of the antibody targetingthe tumor cell antigen, such as the anti-CD19, CD20, and CD33 antigens,in combination with another Fab region recognizing CD16 on NK cell leadsto stimulation of the NK cells followed by tumor cell killing.

In some embodiments, the additional therapeutic agent comprises one ormore chemotherapeutic agents or a radioactive moiety. Chemotherapeuticagent refers to cytotoxic antineoplastic agents, that is, chemicalagents which preferentially kill neoplastic cells or disrupt the cellcycle of rapidly-proliferating cells, or which are found to eradicatestem cancer cells, and which are used therapeutically to prevent orreduce the growth of neoplastic cells. Chemotherapeutic agents are alsosometimes referred to as antineoplastic or cytotoxic drugs or agents,and are well known in the art.

In some embodiments, the chemotherapeutic agent comprises ananthracycline, an alkylating agent, an alkyl sulfonate, an aziridine, anethylenimine, a methylmelamine, a nitrogen mustard, a nitrosourea, anantibiotic, an antimetabolite, a folic acid analog, a purine analog, apyrimidine analog, an enzyme, a podophyllotoxin, a platinum-containingagent, an interferon, and an interleukin. Exemplary chemotherapeuticagents include, but are not limited to, alkylating agents(cyclophosphamide, mechlorethamine, mephalin, chlorambucil,heamethylmelamine, thiotepa, busulfan, carmustine, lomustine,semustine), animetabolites (methotrexate, fluorouracil, floxuridine,cytarabine, 6-mercaptopurine, thioguanine, pentostatin), vinca alkaloids(vincristine, vinblastine, vindesine), epipodophyllotoxins (etoposide,etoposide orthoquinone, and teniposide), antibiotics (daunorubicin,doxorubicin, mitoxantrone, bisanthrene, actinomycin D, plicamycin,puromycin, and gramicidine D), paclitaxel, colchicine, cytochalasin B,emetine, maytansine, and amsacrine. Additional agents includeaminglutethimide, cisplatin, carboplatin, mitomycin, altretamine,cyclophosphamide, lomustine (CCNU), carmustine (BCNU), irinotecan(CPT-11), alemtuzamab, altretamine, anastrozole, L-asparaginase,azacitidine, bevacizumab, bexarotene, bleomycin, bortezomib, busulfan,calusterone, capecitabine, celecoxib, cetuximab, cladribine,clofurabine, cytarabine, dacarbazine, denileukin diftitox,diethlstilbestrol, docetaxel, dromostanolone, epirubicin, erlotinib,estramustine, etoposide, ethinyl estradiol, exemestane, floxuridine,5-flourouracil, fludarabine, flutamide, fulvestrant, gefitinib,gemcitabine, goserelin, hydroxyurea, ibritumomab, idarubicin,ifosfamide, imatinib, interferon alpha (2a, 2b), irinotecan, letrozole,leucovorin, leuprolide, levamisole, meclorethamine, megestrol,melphalin, mercaptopurine, methotrexate, methoxsalen, mitomycin C,mitotane, mitoxantrone, nandrolone, nofetumomab, oxaliplatin,paclitaxel, pamidronate, pemetrexed, pegademase, pegasparagase,pentostatin, pipobroman, plicamycin, polifeprosan, porfimer,procarbazine, quinacrine, rituximab, sargramostim, streptozocin,tamoxifen, temozolomide, teniposide, testolactone, thioguanine,thiotepa, topetecan, toremifene, tositumomab, trastuzumab, tretinoin,uracil mustard, valrubicin, vinorelbine, and zoledronate. Other suitableagents are those that are approved for human use, including those thatwill be approved, as chemotherapeutics or radiotherapeutics, and knownin the art. Such agents can be referenced through any of a number ofstandard physicians' and oncologists' references (e.g. Goodman &Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition,McGraw-Hill, N.Y, 1995) or through the National Cancer Institute website(fda.gov/cder/cancer/druglistfrarne.htm), both as updated from time totime.

Immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide, andpomalidomide stimulate both NK cells and T cells. As provided herein,IMiDs may be used with the modulated therapeutic immune cells for cancertreatments.

A variety of diseases may be ameliorated by introducing the cells of theinvention to a subject suitable for adoptive cell therapy. Examples ofdiseases including various autoimmune disorders, including but notlimited to, alopecia areata, autoimmune hemolytic anemia, autoimmunehepatitis, dermatomyositis, diabetes (type 1), some forms of juvenileidiopathic arthritis, glomerulonephritis, Graves' disease,Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, myastheniagravis, some forms of myocarditis, multiple sclerosis,pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa,polymyositis, primary biliary cirrhosis, psoriasis, rheumatoidarthritis, scleroderma/systemic sclerosis, Sjögren's syndrome, systemiclupus, erythematosus, some forms of thyroiditis, some forms of uveitis,vitiligo, granulomatosis with polyangiitis (Wegener's); hematologicalmalignancies, including but not limited to, acute and chronic leukemias,lymphomas, multiple myeloma and myelodysplastic syndromes; solid tumors,including but not limited to, tumor of the brain, prostate, breast,lung, colon, uterus, skin, liver, bone, pancreas, ovary, testes,bladder, kidney, head, neck, stomach, cervix, rectum, larynx, oresophagus; and infections, including but not limited to, HIV—(humanimmunodeficiency virus), RSV—(Respiratory Syncytial Virus),EBV—(Epstein-Barr virus), CMV—(cytomegalovirus), adenovirus- and BKpolyomavirus-associated disorders.

EXAMPLES

The following examples are offered by way of illustration and not by wayof limitation.

Example 1—Methods and Materials

In Vitro T Cell Culture. Fresh leukopaks (AllCells, Alameda, Calif.)were obtained from healthy donors, from which T cells were negativelyselected using the EasySep Human T Cell Enrichment Kit (Stem CellTechnologies, Vancouver, Canada). The freshly isolated T cells werealiquoted and cryopreserved. On the day the screens were initiated, Tcells were thawed and washed into X-Vivo 15 with 5% human AB serum,IL-2, pen/strep, and additional supplements. Cells were dispensed intoflat-bottom 384-well plates at 5×10⁵ cells/ml with anti-CD3/Anti-CD28dynabeads (ThermoFisher, Waltham, Mass.) at a 3:1 bead-to-cell ratio.Individual compounds were added at a final concentration of 10 μM toeach well from column 3 to column 22 of each plate. Positive andnegative controls were added to additional wells. Cells were incubatedfor about 6 days at 37 degrees with 5% CO2.

Flow Cytometry. On Day 6 of culture, cells were stained with a fixableviability marker and fluorophore-conjugated antibodies: CD3, CD4, CD8,CD45RA, CD45RO, CD62L, CCR7, CD27, and CD122 (BD Biosciences, San Jose,Calif.; and BioLegend, San Diego, Calif.). Fluorescent absolute countingbeads (Spherotech, Lake Forest, Ill.) were added just prior toacquisition. Data acquisition was performed on a BD Fortessa X-20 (BDBiosciences) and data were analyzed using Treestar software (FlowJo,Ashland, Oreg.) and Spotfire (Tibco, Boston, Mass.).

Culturing cells in large-scale for phenotypic and exhaustion markerevaluation. Isolated CD8 T cells were activated in bulk on day 0 usingCTS (Cell Therapy Systems) Dynabeads™ CD3/CD28 (Thermo FisherScientific, Waltham, Mass.) in T cell media supplemented with IL-2. Onday 1, cells were transduced with the CAR Construct and cell density wasadjusted to 0.5×10⁶/ml and 10⁶ cells were seeded into 12-well plates inthe presence of vehicle, TWS119, or DCC-2036. On day 4, cells weretransferred into 6-well plates and 2 ml of T cell media was added toeach well. On day 6 another 2 ml of media was added to each well. On day8, CAR T cells were analyzed on a flow cytometer for surface expressionof phenotypic markers (CD62L, CCR7, and CD27) and exhaustion markers(PD-1 and Tim-3).

Example 2—Agent for Immune Cell Modulation

Data were analyzed to identify compounds that either produced a higherproportion or greater absolute number of phenotypically identifiednaïve, stem cell memory, or central memory T cells. These cells arecharacterized by expression of CCR7 and CD62L. Therefore, cellsco-expressing both of these identifying markers were evaluated. Withinthe viable CD4+ population and viable CD8+ population, the percent ofcells co-expressing CCR7 and CD62L was determined. The expression ofeither CD62L or CCR7 on T cells, as indicative of the desired T cellsubsets, have been described as having favorable functionalcharacteristics for CAR-T cell therapy, and potentially other adoptive Tcell therapies. Under the treatment of dorsomorphin, heptelidic acid,GSK3 inhibitor, 6-Mercaptopurine, AC-93253 iodide, tiratricol, PI-103,5-Azacytidine, 5,7-dichloro-8-Quinolinol, Nitrofurantoin,5-chloro-7-iodo-8-Quinolinol, or diethylenetriaminepentaacetic acid, thenumber or ratio of cells co-expressing CCR7 and CD62L increased in bothviable CD4+ population and viable CD8+ population (Table 2). Under thetreatment of fulvestrant, thapsigargin, SU 4312, fludarabine,2-Naphthacenecarboxamide,7-chloro-4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahy, nifuroxazide,edrophonium chloride, the number or ratio of cells co-expressing CCR7and CD62L increased at least in viable CD8+ population (Table 2). Underthe treatment of 1-Pyrrolidinecarbodithioic acid, ammonium salt, U0126,telmisartan, cyclosporin A,1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, BAY 61-3606,Protoporphyrin IX disodium, rapamycin, roscovitine, PAC-1, tosufloxacinhydrochloride, BIX01294, and terfenadine, the number or ratio of cellsco-expressing CCR7 and CD62L increased at least in viable CD8+population (Table 2).

In addition, GSK3 (Glycogen synthase kinase 3) inhibitor was shown topreserve CD3-CD19-CD56+NK cells, and increased the adaptive NK cellsubpopulation by affecting cell maturation and subtype skewing, based onobservation of including, but not limited to CD57+ and NKG2C+expression.

The number of events in each of these gates relative to the number ofabsolute counting beads in each sample was calculated, defining arelative measure of the absolute number of naïve, stem cell memory, orcentral memory T cells within the CD4+ and/or CD8+ populations. Az-Score relative to the screened compound samples within each 384-wellplate was calculated for each of these four values: 1) percentCCR7+CD62L+ in CD4+, 2) percent CCR7+CD62L+ in CD8+, 3) absoluterelative number of CCR7+CD62L+ in CD4+, and 4) absolute relative numberof CCR7+CD62L+ in CD8+(FIGS. 1A and 1 i). Z-scores were also calculatedfor the percent viability of all cells within each sample and therelative absolute number of cells within each sample. A “Z-Score” is astatistical measurement of a score's relationship to the mean in a groupof scores. A Z-score of 0 means the score is the same as the mean. AZ-score can also be positive or negative, indicating whether it is aboveor below the mean and by how many standard deviations.

Eliminating compounds that have a detrimental impact on T cellproliferation or viability, focuses efforts on compounds that are mostlikely to be amenable to T cell manufacturing strategies. Primary hitcompounds were selected by the following criteria: ‘percent viability’Z-score of greater than −1, ‘relative absolute number of cells’ Z-scoreof greater than −1, and Z-score of one of the 4 values of greater than+2. 34 compounds (Table 2) were selected for having much higher Z-Scoresand meeting the above criteria for more than 1 of the 4 primary values.An additional 5 compounds are also included for their abilities tomodulate T cells (Table 3)

TABLE 2 Agents for T Cell Modulation in Adoptive Cell Therapies CD8 CD4Compounds CAS Number Compound Information Group Group Descriptor Hit HitDorsomorphin 866405-64-3 AMPK inhibitor I Metabolism & CD8 CD4 NutrientSensing Heptelidic acid 74310-84-2 GAPDH inhibitor I Metabolism & CD8CD4 Nutrient Sensing 1-Pyrrolidinecarbodithioic 5108-96-3 Preventsinduction of I Metabolism & CD4 acid, ammonium salt nitric oxidesynthetase Nutrient Sensing GSK3 Inhibitor For example-BIO; GSK-3α/βinhibitor II Signaling Pathways CD8 CD4 667463-62-9 6-Mercaptopurine6112-76-1 Competes with purine II Signaling Pathways CD8 CD4 derivativeshypoxanthine and guanine for enzyme HGPRT AC-93253 iodide 108527-83-9Subtype selective II Signaling Pathways CD8 CD4 RAR (RARα) agonistTiratricol 51-24-1 Thyroid hormone analogue II Signaling Pathways CD8CD4 PI-103 371935-74-9 mTOR/PI3K inhibitor II Signaling Pathways CD8 CD4Fulvestrant 129453-61-8 Estrogen receptor II Signaling Pathways CD8antagonist Thapsigargin 67526-95-8 sarco/ER Ca2+- II Signaling PathwaysCD8 ATPase antagonist SU 4312 5812-07-7 VEGF receptor protein IISignaling Pathways CD8 tyrosine kinase 1/2 and PDGF receptor inhibitorU0126 109511-58-2 MAPK/ERK kinase; II Signaling Pathways CD4 antagonizesAP-1 transcriptional activity Telmisartan 144701-48-4 Micardis;angiotensin II Signaling Pathways CD4 II receptor anatagonistCyclosporin A 59865-13-3 Neoral; immunosuppressive II Signaling PathwaysCD4 1,3,5-tris(4-hydroxyphenyl)- 263717-53-9 PPT; a specific IISignaling Pathways CD4 4-propyl-1H-pyrazole estrogen receptor α (ERα)agonist BAY 61-3606 732983-37-8 Spleen tyrosine kinase II SignalingPathways CD4 (Syk) inhibitor Protoporphyrin IX disodium 553-12-8 GCS(guanylate cyclase) II Signaling Pathways CD4 activator rapamycin53123-88-9 Sirolimus; II Signaling Pathways CD4 immunosuppressant5-Azacytidine 320-67-2 Cytosine nucleoside III Proliferation and CD8 CD4analog that interferes Apoptosis with nucleic acid synthesis Fludarabine21679-14-1 Purine analog that III Proliferation and CD8 interferes withApoptosis nucleic acid synthesis Roscovitine, (S)-Isomer 186692-45-5Cyclin-dependent III Proliferation and CD4 kinase (Cdk) inhibitorApoptosis PAC-1 315183-21-2 Procaspase-3 III Proliferation and CD4activating compound; Apoptosis 8-Quinolinol,5,7-dichloro- 773-76-2Capitrol; Antibiotic IV Anti-infective CD8 CD4 Nitrofurantoin 67-20-9Antibiotic IV Anti-infective CD8 CD4 8-Quinolinol,5-chloro-7-iodo-130-26-7 Clioquinol; Antibiotic IV Anti-infective CD8 CD42-Naphthacenecarboxamide, 64-73-3 Ribosomal protein IV Anti-infectiveCD8 7-chloro-4-dimethylamino)- synthesis inhibitor1,4,4a,5,5a,6,11,12a-octahy Nifuroxazide 965-52-6 Nitrofuran antibioticIV Anti-infective CD8 Tosufloxacin hydrochloride 100490-36-6 Ozex;Fluoroquinolone IV Anti-infective CD4 antibiotic Sertraline 79617-96-2Zoloft; antidepressant V Other CD8 CD4 Diethylenetriaminepentaacetic67-43-6 Iron chelating agent V Other CD8 CD4 acid, penta sodiumEdrophonium chloride 116-38-1 Reversible V Other CD8acetylcholinesterase inhibitor BIX01294 1392399-03-9 GLP and G9ahistonelysine V Other CD4 methyltransferase inhibitor Terfenadine50679-08-8 Antihistamine V Other CD4 dmPGE2 39746-25-3 Prostaglandinmolecule V Other

TABLE 3 Additional Agents for T Cell Modulation in Adoptive CellTherapies CAS Compound Group Compounds Number Information GroupDescriptor 2-DG 154-17-6 Inhibits glycolysis I Metabolism & NutrientSensing GSK3 For GSK3 inhibitor II Signaling Inhibitor example- PathwaysTWS119: 601514- 19-6 HS173 1276110- PI3K inhibitor II Signaling 06-5Pathways LY294002 154447- PI3K inhibitor II Signaling 36-6 PathwaysPictilisib 957054- PI3K inhibitor II Signaling 30-7 Pathways

Example 3—In Vitro Triage Experiments of the Selected Compounds

In vitro experiments are performed to optimize methods for compoundexposure and triage compounds that have detrimental impacts on T cellfunctions. Initial tests determine optimal dose of individual compoundswhile also evaluating whether the impact on naïve, stem cell memory, andcentral memory T cells observed previously are replicated in additionaldonors. To triage compounds with potential detrimental functionalimpacts on T cells, in vitro assessments for proliferative capacity,ability to polarize to Th1 and Th17, survival through acryopreservation/thaw cycle, transduction efficiency, and tumoricidalactivity of CAR-transduced T cells are performed. Compounds thatreproducibly improve ratio or number of naïve, stem cell memory, orcentral memory T cells during expansion without significant negativeimpacts on T cell function are tested in combination and assessed foradditive or synergistic effects. Through these assessments, leadcandidates or combinations are prioritized for additional testing invivo.

Example 4—In Vivo Models of Adoptive Cellular Therapy Using the SelectedCompounds

To translate the results of the in vitro screening and follow up invitro triage experiments the lead candidates of the selected compoundsare applied to in vivo models of adoptive cellular therapy.Specifically, the impact of small molecule modulation is interrogated onadoptive cellular therapy in regards to engraftment, tumoricidalactivity, secondary tumoricidal responses, migration, cellularpersistence, and graft-versus-host disease. Other readouts which arehall marks of durable adoptive cellular therapy that have been found tocorrelate with efficacious responses in the clinic are alsointerrogated.

These experiments are conducted either in a humanized system, in whichhuman cells are adoptively transferred into immuno-deficient NSG micebearing human tumors, or in a surrogate murine model, in which animmuno-competent animal bears a syngeneic tumor and is treated withsyngeneic cellular therapy.

In either the surrogate or humanized model system, mice are injectedwith a luciferized lymphoma or other tumor of interest. Soon thereafter,the adoptive cellular therapy which has been pre-treated with vehicle ormodulating compounds disclosed herein is administered. Dose of both thecell therapy and tumor is optimized to enable a window in which positiveor detrimental effects of the compound treatment can be observed. Theanimal weight, plasma cytokine concentrations, abundance of tumor andadoptive cellular therapy in peripheral blood, secondary lymphoid organsand tumor mass; tumor burden, tumor metastasis, and phenotype of thecellular therapy are monitored for the duration of the experiment.

Compounds that are able to ameliorate one or many tumor-relatedparameters in vivo have expected effects including, but not limited to,decreasing the cellular therapy dose required for effective tumorclearance, increasing the persistence of adoptive cellular therapy inthe peripheral blood, enhancing migration to tumor sites, and/orincreased survival against challenge with high tumor dose.

Example 5—Synergistic Increase in CD27 with Rapa+dmPGE2 Treatment in TCells

CD27 is a member of the TRA-linked TNF (Tumor Necrosis Factor) receptorfamily that also includes 4-1BB and OX-40. These transmembrane proteinsare involved in the regulation of lymphocyte function. In humans, mostnaïve peripheral T cells (Tn) express CD27. Once the naïve peripheral Tcells are activated, the expression of CD27 is significantly increased.However, terminal effector differentiation of T cell is associated withirreversible loss of CD27 (Hintzen et al. 1994). Further elucidation ofthe role of CD27 demonstrated that it was required for the generationand long-term maintenance of T cell immunity (Hendriks et al. 2000).

Cells treated with rapamycin (referred to as “rapa” from time to time),dimethyl prostaglandin E2 (dmPGE2) or a combination of both compoundsexhibited significant differences in many aspects when compared to cellstreated with vehicle alone (DMSO). However, one of the strongest changesobserved was a synergistic increase in the level of CD27 expressed onthe surface of CD8 T cells treated with rapa+dmPGE2 relative to theexpression of CD27 on CD8 T cells treated with vehicle or eithercompound alone. In order to perform these studies, isolated CD8 T cellswere activated in bulk on day 0 using CTS (Cell Therapy Systems)Dynabeads™ CD3/CD28 (Thermo Fisher Scientific, Waltham, Mass.) in T cellmedia supplemented with IL-2. On day 1, cells were transduced with theCAR Construct 1 shown in FIG. 2 and cell density was adjusted to0.5×10⁶/ml and 10⁶ cells were seeded into 12 well plates in the presenceof vehicle, 20 nM rapa, 10 μM dmPGE2 or both compounds at the sameconcentrations. On day 4, cells were transferred into 6-well plates and2 ml of media was added to each well. On day 6 another 2 ml of media wasadded to each well. On day 8, cells were analyzed on a flow cytometerfor cell surface expression of CD27.

The expression of CD27 in treated and untreated T cells from twoindependent donors is shown in FIG. 3 . The expression of CD27 observedwith vehicle treatment was defined as an MFI (mean fluorescenceintensity) of 1 and the relative MFI of cells treated with compound wascalculated. When CD8 T cells are incubated with rapa alone, the CD27expression increases approximately 2-fold; with dmPGE2 alone there is noincrease, i.e., dmPGE2 alone has no impact on the CD27 expression.However, when CD8 T cells are incubated in the presence of a combinationof rapamycin and dmPGE2 (rapa+dmPGE2), the increase in CD27 expressionrelative to the vehicle is almost 6-fold, a level that cannot beexplained simply through additive effects of the two individualcompounds. Thus, it appears that the cell surface expression of CD27induced by the compound combination is synergistic; and the compoundcombination appears to drive for the phenotype of naïve T cells ratherthan T effector cells.

Example 6—Rapa+dmPGE2 Treatment Increases the T Cell Central MemorySubset

Studies in both non-human primate and NOD/Scid IL-2RγC^(null) (NSG)mouse models have demonstrated that T cells with a central memory (Tcm)phenotype have improved persistence after adoptive transfer (Berger etal. 2008; Wang et al. 2011). In addition, CAR-expressing CD4 and CD8central memory T cell (Tcm) subsets were administered to Non-Hodgkinlymphoma patients after hematopoietic stem cell transplantation, and theTcm-derived CAR-T cells demonstrated improved expansion, indicating thatTcm may have a therapeutic advantage in treatment of human cancers. Thecapability of the rapamycin and dmPGE2 combination in skewing thepopulation toward a Tcm phenotype was accessed. The CD8 Tcm subset wasdefined based on the expression of the cell surface markers CD45RA andCCR7. FIG. 4A shows a scatter plot of the CD45RA and CCR7 expression inT cells that have not been activated, which was then used for gating ofthe T cell subsets including Tcm, naïve (Tn), effector memory (Tem) andCD45RA+ effector memory (Temra) cells. Tn and Tcm cells are the leastdifferentiated and have the greatest proliferative potential while Temracells are the most fully differentiated, have poor proliferativepotential but strong effector function (D'Asaro et al. 2006).

CD8 T cells were treated with vehicle, rapa, dmPGE2 or rapa+dmPGE2 asdescribed in Example 5. FIG. 4B shows a schematic representation of theT cells subsets present in cultures after respective treatment. FIG. 4Cshows the flow cytometry analysis used to identify the differentsubsets. Relative to vehicle or either compound alone, there is anincrease in the percentage of Tcm subset accompanied by a loss in themore differentiated Tem subset after treatment with the combination ofrapamycin and dmPGE2. Thus, treatment with rapa+dmPGE2 causes anincrease in a more desirable T cell subset (Tcm) for CAR-T cell therapy.Without being limited by theory, the phenotype shifting from Tem to Tcm,and/or the promoted Tcm expansion may have contributed to the increasedpercentage of Tcm in the modulated population.

Example 7—Rapa+dmPGE2 Treatment Reduces T Cell Exhaustion MarkerExpression

T cell dysfunction due to ‘exhaustion’ is a state that can precludeadequate control of cancer or infection. T cell exhaustion ischaracterized by poor effector cell function and increased expression ofmultiple cell surface proteins, collectively known as exhaustionmarkers, including PD-1 and Tim-3 (Wherry and Kurachi 2015). Todetermine the effect of compound treatment of CAR-T cells on exhaustionmarker expression, cells from two different donors were prepared andtreated as described in Example 5 then stained for PD-1 and Tim-3 andexpression was determined using flow cytometry. As shown in FIG. 5 ,treatment with either rapamycin or dmPGE2 decreased PD-1 expressionwhile treatment with rapamycin and dmPGE2 combination led to a slightlygreater reduction in PD-1 expression relative to vehicle, or eachindividual compound treatment. As to Tim-3 expression under varioustreatments, rapamycin alone decreased Tim-3 expression while dmPGE2alone showed no effect when compared to vehicle. However, thecombination of rapamycin and dmPGE2 reduced Tim-3 expression moremarkedly as compared to any single compound treatments or no treatment.An enhanced reduction in Tim-3 expression by rapa+dmPGE2 combination wasobserved relative to vehicle, rapamycin, and dmPGE2, despite the factthat, when used alone, dmPGE2 has no effect on Tim-3 expression. Thisdata indicated that treating T cells with rapamycin and dmPGE2combination enhances the cell's anti-tumor capability by reducing T cellexhaustion that contributes to immune dysfunction.

Example 8—Rapa+dmPGE2 Treatment Increases T Cell Mitochondrial SpareRespiratory Capacity

Cells generally utilize two major energy pathways, glycolysis andmitochondrial respiration. It has been shown that mitochondrial sparerespiratory capacity (SRC), which is the extra capacity available incells to produce energy in response to increased stress or work, isincreased in T memory cells but not in T effector cells, such as Temra(van der Windt et al. 2012).

We determined whether compound treatment had an effect on SRC usingcells transduced and treated as described in Example 5. After treatment,oxygen consumption rate (OCR), a measure of mitochondrial respirationwas determined using a Seahorse™ XFe96 system (Seahorse Bioscience,Santa Clara, Calif.). To do this, the cells were washed and resuspendedin non-buffered assay medium. The cells were then seeded in 96-wellassay plates, and subjected to the Seahorse™ mitostress test assay. SRCis determined based on the difference between basal OCR and OCR at themaximal respiration rate. FIG. 6 shows that SRC is increased almosttwo-fold in cells treated with rapa+dmPGE2, whereas treatment with eachindividual compound shows only a modest increase of about 20-50%relative to vehicle controls. These data indicate that CAR-T cellstreated with rapa+dmPGE2 not only increase, or being skewed towards, Tcmphenotype, but also demonstrate the desired metabolic profile related tothe memory T cell subset.

Example 9—Genome-Wide Expression Characterization of CD8+ T-CellsTreated with Rapa+dmPGE2

To characterize the genome-wide impact of rapamycin and dmPGE2 treatmentindividually or in combination during the in vitro T cell expansionprocess, RNA from cells that are transduced and treated as described inExample 5 was extracted and analyzed on Human Transcriptome Array gene(microarray) chips (Affymetrix, Santa Clara, Calif.), and the resultswere compared to a vehicle treated sample. FIG. 7A depicts thegenome-wide transcription profiling using microarray chips to show thetranscriptional changes of gene probes induced by the small moleculesindividually and in combination. As shown schematically in FIG. 7B, itis notable that the total number of genes that are up-regulated morethan 2 fold compared to vehicle control under the rapa+dmPGE2 treatmentis 377, whereas this number is 215 and 71 under the treatment ofrapamycin only and dmPGE2 only, respectively. Among the 377 upregulatedgenes, 264 genes are uniquely upregulated by the combination treatment,but not by either individual compound treatment. As to thedown-regulated genes, a total of 581 genes are down-regulated more than2 fold compared to vehicle control under the rapa+dmPGE2 treatment;whereas only 284 and 38 genes are down-regulated under the rapamycinonly and dmPGE2 only treatment, respectively. Among the 581down-regulated genes, 351 genes are uniquely down-regulated by the combotreatment, but not by either individual compound treatment. Thedifferential gene expression profile under single or combinationalcompound treatment demonstrates that the combined treatment using bothsmall molecules has a profound transcriptional effect which issignificantly greater than the sum of each individual treatment,indicating a synergistic response in the presence of both pathwaymodulators.

Some exemplary key genes relevant to T cell function (such as cytokinesecretion, cytotoxicity, exhaustion, costimulation), differentiation(such as cell development, maturation, and memory cell phenotype) andmetabolism (such as glycolysis, amino acid metabolism, cellproliferation) that are differentially expressed under the single orcombinational compound treatments are shown in FIG. 8A, and Table 4.

TABLE 4 Relevant genes that are differentially expressed under thesingle or combinatorial compound treatments UniProtKB/ Swiss-Prot EntryGene Protein Name Identifier 1 TCF7 Transcription factor 7 P36402 2 CD27CD27 antigen P26842 3 LEF1 Lymphoid enhancer- P27782 binding factor 1 4FOXP1 Forkhead box protein P1 Q9H334 5 CCR7 C-C chemokine P32248receptor type 7 6 CREB1 Cyclic AMP-responsive P16220 element-bindingprotein 1 7 CROT Peroxisomal carnitine Q9UKG9 O-octanoyltransferase 8MEF2A Myocyte-specific Q02078 enhancer factor 2A 9 ACACA5′-AMP-activated protein Q13131 kinase catalytic subunit alpha-1 10ALDOA Fructose-bisphosphate P04075 aldolase A 11 ALDOCFructose-bisphosphate P09972 aldolase C 12 CD93 Complement componentQ9NPY3 C1q receptor 13 PGAM1 Phosphoglycerate mutase 1 P18669 14 PGK1Phosphoglycerate kinase 1 P00558 15 LDHA L-lactate dehydrogenase AP00338 chain 16 PDCD1 Programmed cell death 1 Q9NZQ7 ligand 1 17 ENO2Gamma-enolase P09104 18 HK1 Hexokinase-1 P19367 19 IL15 Interleukin-15P40933 20 SMAD7 Mothers against O15105 decapentaplegic homolog 7 21 ELF4ETS-related transcription Q99607 factor Elf-4 22 CD244 Natural killercell Q9BZW8 receptor 2B4 23 GFI1 Zinc finger protein Gfi-1 Q99684 24KLRF1 Killer cell lectin-like Q9NZS2 receptor subfamily F member 1 25RUNX3 Runt-related transcription Q13761 factor 3 26 HK2 Hexokinase-2P52789 27 PFKM ATP-dependent 6- P08237 phosphofructokinase 28 CD74 HLAclass II P04233 histocompatibility antigen gamma chain 29 LAG3Lymphocyte activation P18627 gene 3 protein 30 CD69 Early activationantigen Q07108 CD69 31 CD58 Lymphocyte function- P19256 associatedantigen 3 32 PXN Paxillin P49023 33 Tim-3 T-cell immunoglobulin andQ8TDQ0 mucin domain-containing protein 3T cell immunoglobulin mucin 34CYP1A1 Cytochrome P450 1A1 P04798 35 KIR3DL2 Killer cell immunoglobulin-P43630 like receptor 3DL2 36 TNFRSF9 Tumor necrosis factor Q07011receptor superfamily member 9 37 FAS Tumor necrosis factor P25445receptor superfamily member 6 38 SLC2A3 Solute carrier family 2, P11169facilitated glucose transporter member 3 39 SLC1A5 Neutral amino acidtransporter Q15758 B 40 GPR56 Adhesion G-protein coupled Q9Y653 receptorG1 41 PRDM1 PR domain zinc finger O75626 protein 1 42 IL7R Interleukin-7receptor P16871 subunit alpha 43 SLC3A2 4F2 cell-surface antigen P08195heavy chain 44 PRF1 PRF1 P14222 45 IL2 Interleukin-2 P60568 46 NKG7Protein NKG7 Q16617 47 SLC7A5 Large neutral amino acids Q01650transporter small subunit 1 48 SLAMF7 SLAM family member 7 Q9NQ25 49GZMH Granzyme H P20718 50 TNF Tumor necrosis factor P01375 51 CCL4 C-Cmotif chemokine 4 P13236 52 IFNG Interferon gamma P01579

Example 10—Gene Panel Characterization of CD8+1-Cells Treated withRapa+dmPGE2

To further characterize the impact of both small molecules incombination on the expression changes for a panel of genes important forT cell maturation, total RNA was extracted from cells transduced andprepared as in Example 5. The RNA was analyzed on Affymetrix humantranscriptome arrays and fold changes (relative to vehicle treatment) ofthe genes in the panel were determined. The inclusion of both smallmolecules had a profound transcriptional impact on many key T cell genessome of which are known to promote a memory phenotype as well impactingmetabolism (FIG. 8A). Treatment with both rapamycin and dmPGE2 resultedin synergistic effects for both transcription factor genes (FIG. 8B),and genes involved in glycolysis (FIG. 8B). The transcription factorsTCF7 and LEF1 are important for the generation of functional memorycells (Zhou et al., J Immunol. 2012; 189(6): 2722-2726), and treatmentwith both rapamycin and dmPGE2 increased the expression of these genesmore than either compound alone (FIG. 8B). BLIMP-1 is a transcriptionalrepressor gene expressed in terminally differentiated and effectormemory T cells, and BLIMP-1 deficiency promotes the acquisition ofcentral memory T cell properties (Rutishauser et al., Immunity. 2009;31(2): 296-308). Treatment with the combination of rapamycin and dmPGE2resulted in a greater decrease of BLIMP-1 expression compared to eithercompound alone (FIG. 8B). Interestingly, BLIMP-1 is slightly upregulatedwhen treated with dmPGE2 alone, which is consistent with the observationthat dmPGE2 promotes effector T cell differentiation (Sreeramkumar etal., 2015). So it is surprising that the combination treatment usingboth rapamycin and dmPGE2 not only “corrects” dmPGE2's tendency indriving T cell differentiation, but also achieves a synergistic effectwhen combined with rapamycin in down-regulating BLIMP-1 expression.

In addition to changes in transcription factor expression, thecombination of rapamycin and dmPGE2 also affected the expression ofgenes involved in metabolism. The genes ALDOC, ENO2, and PGK1 are allinvolved in different steps of the glycolysis pathway, and the combinedtreatment with rapamycin and dmPGE2 resulted in down-regulation of thesegenes (FIG. 8C). In contrast, treatment with either compound alonecaused an up-regulation of all three of these glycolysis-related genes(FIG. 8C). Activated T cells preferentially switch to aerobic glycolysiswhich is more suited for supporting full effector function (Chang etal., Cell. 2013; 153(6): 1239-1251). Induction of high glycolyticactivity in CD8+ T cells drives T cells toward a terminallydifferentiated state rather than memory cells (Sukumar et al., J ClinInvest. 2013; 123(10): 4479-4488). Areduction in the ability of cellstreated with rapamycin and dmPGE2 to undergo glycolysis through reducedexpression of ALDOC, ENO2, and PGK1, along with the increase in sparerespiratory capacity as shown in Example 8, supports the notion that thecombination treatment is skewing the cell towards a central memoryphenotype.

Example 11—RT-qPCR Quantitation of CCR7 and CD62L in CD8+ T-CellsTreated with Rapa+dmPGE2

To further characterize the impact of rapa+dmPGE2 in memory cellskewing, the expression changes for two key genes, CCR7 and CD62L, knownto be highly expressed in memory T cells were investigated. The totalRNA was extracted from cells treated with compounds as described inExample 5. The RNA was analyzed using RT-qPCR and TaqMan gene expressionassays specific to each gene. Relative quantitation of each genetranscript was determined for each in vitro treatment. FIGS. 9A and 9Bshow the dramatic increase in expression of both key memory T cell geneswith rapa+dmPGE2 treatment, while the individual compounds inducedrelatively minimal transcriptional increases for both of these genes.FIG. 9C showed a similar effect of rapa+dmPGE2 treatment on CD27 geneexpression, which corroborates the cell surface expression of CD27 inExample 5 and FIG. 3 . The observed requirement for both rapamycin anddmPGE2 implies a synergistic response between pathways to promote theexpressional changes towards a memory T cell.

Example 12—Treatment with Rapa+dmPGE2 Improves CAR-T Cell Expansion in aSerial Killing Assay

A critical prognostic marker of CAR-T cell therapy in clinical trials isthe expansion of the CAR-T cells in the patient post-treatment (Porteret al. 2015). An in vitro serial killing assay, where CAR-T cells areallowed to “clear” tumor cells in vitro over multiple rounds, is a modelthat can assess CAR-T expansion in the presence of tumor cells thatexpress the antigen recognized by the CAR. To this end, CD8 T cells weretransduced to obtain CAR-T cells, and treated as in Example 5, thencryopreserved. After thawing, the CAR-T cells were co-cultured withNalm6 tumor cells that express endogenous CD19 and transgenic mKate2, afar-red fluorescent protein. Expression of eGFP in the CAR-T cells andmKate2 in the Nalm6 target cells allows easy and reliable identificationand counting of both cell types in the serial killing assay. Prior tostarting each round of killing, cell numbers were adjusted so that thesame ratio of CAR-T cell to Nalm6 target cell was maintained across theCAR-T cell cultures generated from different compound treatments.

FIG. 10A shows the fold expansion of CAR-T cells from two donors aftereach round of tumor cell killing. CAR-T cells treated with rapa+dmPGE2show consistently improved expansion relative to vehicle or eithercompound individually. When total expansion over all three rounds ofserial killing is determined (FIG. 10B), rapa+dmPGE2 shows much greaterexpansion than either compound individually indicating a potentialsynergistic effect in this critical parameter. Despite the observeddifference in expansion, the ability of the CAR-T cells to kill tumorcells in vitro was unaffected for three rounds of in vitro killing.CAR-T cells previously treated with rapa, dmPGE2, or rapa+dmPGE2successfully cleared target cells over time (FIG. 11A). At the end ofround three, flow cytometry analysis of viable cells in the co-culturesshow very few mKate2 positive cells for vehicle and all compound-treatedcells (FIG. 11B).

Example 13—Demonstration of Improved CAR-T Cell Expansion withRapa+dmPGE2 in Larger-Scale Culture Format

To determine the effect of rapa+dmPGE2 on CD4 T cells and to demonstratethat the increased expansion of rapa+dmPGE2 would scale to largerexpansion formats, CD4 and CD8 T cells were separately activated andexpanded in vitro. The cells were transduced with the CAR-2 construct(FIG. 2 ) one day after activation and then replated into 24-well GREXplates with or without compounds. Half the media was replaced every twodays thereafter until harvest one week post activation. FIG. 12demonstrates that the addition of rapa+dmPGE2 results in increased CD4and CD8 T cell expansion (FIG. 12A) and viability (FIG. 12B) at one weekpost activation relative to DMSO.

Example 14—Improved In Vivo Efficacy of Rapa+dmPGE2-Treated CAR-T Cells

To determine whether rapa+dmPGE2 treatment increased the in vivo tumorclearance and persistence of CAR-T cells NSG mice were injected withNalm-6-luc, a CD19+ human tumor line engineered to express fireflyluciferase. CAR-T cells were generated from CD4+ and CD8+ T cells thatwere separately activated and expanded in vitro. The cells weretransduced with the CAR-2 construct (FIG. 2 ) one day after activationand then replated into 24-well GREX plates with or without thecompounds. Half the media was replaced every two days thereafter untilharvest one week post-activation. One week after tumor injection, theNSG mice were treated with 0.2×10⁶ CD4 and CD8 CAR-T cells mixed at a1:1 ratio via retroorbital injection. The mice were imaged periodicallyto determine tumor burden. FIG. 13A shows that CAR-T cells treated withrapa+dmPGE2 or the PI3K inhibitor PI-103 were able to clear tumor fromthe majority of mice while those treated with untransduced T cells, orCAR-T cells treated with DMSO, U0126 or TWS119, demonstrated minimaltumor control.

Example 15—Improved Secondary Tumor Anti-Tumor Response withRapa+dmPGE2-Treated CAR-T Cells

To determine whether the CAR-T cells that mediated initial tumorclearance were able to persist and provide long-term protection, themice that survived primary tumor challenge (Example 14) wererechallenged with K562-CD19, a human myelogenous leukemia lineengineered to express CD19, 60 days after the primary tumor injection.The only two groups that had mice that survived until the start of thisrechallenge study were the rapa+dmPGE2 and PI-103-treated cohorts. FIG.13B shows that at 21 day post challenge with secondary tumor 75% (¾) ofthe rapa+dmPGE2-treated CAR-T mice and 60% (⅗) of the PI103CAR-T-treated mice had no detectable tumor.

Example 16—Improved In Vivo Efficacy of Cryo-PreservedRapa+dmPGE2-Treated CAR-T Cells

To determine whether rapa+dmPGE2 treatment increased the in vivo tumorclearance and persistence of CAR-T cells after cryopreservation, weinjected NSG mice with 0.5×10⁶ Nalm-6-luc, a CD19 expressing human tumorline that was engineered to express firefly luciferase. Four days latermice were treated with 1.0×10⁶, 0.5×10⁶ or 0.2×10⁶ CD4 and CD8 CAR-Tcells mixed at a 1:1 ratio. CAR-T cells were generated from CD4+ andCD8+ T cells that were separately activated and expanded in vitro asdescribed in Example 14. The cells were transduced with the CAR-2construct (FIG. 2 ) one day after activation and then replated into24-well GREX plates with or without compounds. Half the media wasreplaced every two days thereafter until harvest one week postactivation. Cells were cryopreserved and then thawed for use. NSG micewere infused with tumor cells followed one week later by CAR-T transfervia retroorbital injection.

In one experiment, a suboptimal dose of CAR-T cells (0.2×10⁶) that hadbeen grown in the presence of (1) DMSO; (2) Rapamycin; or (3) Rapamycinand dmPGE₂ (right panel, solid line) were injected into thetumor-bearing mice. CAR-T cells treated with Rapamycin (FIG. 15A) orRapamycin and dmPGE2 (FIG. 15B) were able to reduce tumor burden withina week of adoptive transfer while CAR-T cells treated with DMSO werenot. No mice injected with DMSO-treated CAR-T cells were able to reducetumor burden below detection and all mice failed to have sustainedanti-tumor responses and relapsed (FIGS. 15A and 15B). As shown in FIG.15A, one of four mice injected with Rapamycin-treated CAR-T cellsrelapsed. The other three injected with Rapamycin-treated CAR-T cellswere able to reduce tumor burden below detection, and one of those threemice was able to sustain long-term anti-tumor responses while the othertwo of the three with undetectable tumor burden did not survive beyond30 and 45 days, respectively (FIG. 15A). Among the four mice injectedwith Rapamycin and dmPGE2-treated CAR-T cells, two of the four were ableto sustain long-term anti-tumor responses, while the other two relapsed.One of the two mice which sustained long-term anti-tumor responses wasable to reduce tumor burden below detection. All four mice injected withRapamycin and dmPGE2-treated CAR-T cells survived beyond 60 days.

One skilled in the art would readily appreciate that the methods,compositions, and products described herein are representative ofexemplary embodiments, and not intended as limitations on the scope ofthe invention. It will be readily apparent to one skilled in the artthat varying substitutions and modifications may be made to the presentdisclosure disclosed herein without departing from the scope and spiritof the invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which thepresent disclosure pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated as incorporatedby reference.

The present disclosure illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations that are not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising,”“consisting essentially of,” and “consisting of” may be replaced witheither of the other two terms. The terms and expressions which have beenemployed are used as terms of description and not of limitation, andthere is no intention that in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the present disclosure claimed. Thus, itshould be understood that although the present disclosure has beenspecifically disclosed by preferred embodiments and optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the appended claims.

1.-120. (canceled)
 121. A composition comprising a combination, whereinthe combination comprises: (a) a mammalian target of rapamycin (mTOR)inhibitor; and (b) dimethyl prostaglandin E2 (dmPGE2) or an analogue orderivative thereof.
 122. The composition of claim 121, wherein: (a) themTOR inhibitor is selected from rapamycin, and analogues and derivativesthereof; or (b) the analogue or derivative of dmPGE2 is selected fromthe group consisting of PGE₂, 16,16-dimethyl PGE₂p-(p-acetamidobenzamido) phenyl ester, 11-deoxy-16,16-dimethyl PGE₂,9-deoxy-9-methylene-16, 16-dimethyl PGE₂, 9-deoxy-9-methylene PGE₂,9-keto Fluprostenol, 5-trans PGE₂, 17-phenyl-omega-trinor PGE₂, PGE₂serinol amide, PGE₂ methyl ester, 16-phenyl tetranor PGE₂,15(S)-15-methyl PGE₂, 15(R)-15-methyl PGE₂, 8-iso-15-keto PGE₂, 8-isoPGE₂ isopropyl ester, 8-iso-16-cyclohexyl-tetranor PGE₂, 20-hydroxyPGE₂, 20-ethyl PGE₂, 11-deoxy PGEi, nocloprost, sulprostone, butaprost,15-keto PGE₂, and 19 (R) hydroxy PGE₂.
 123. The composition of claim121, wherein the mTOR inhibitor is selected from the group consisting ofsirolimus, sirolimus derivatives, temsirolimus,40-O-(2-hydroxy)ethyl-rapamycin (everolimus),40-O-(3-hydroxy)propyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, and 40-O-tetrazole-rapamycin.124. The composition of claim 121, wherein: (a) the mTOR inhibitor israpamycin, sirolimus, temsirolimus, 40-O-(2-hydroxy)ethyl-rapamycin(everolimus), 40-O-(3-hydroxy)propyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, or 40-O-tetrazole-rapamycin;and (b) the analogue or derivative of dmPGE2 is PGE₂, 16,16-dimethylPGE₂ p-(p-acetamidobenzamido) phenyl ester, 11-deoxy-16,16-dimethylPGE₂, 9-deoxy-9-methylene-16, 16-dimethyl PGE₂, 9-deoxy-9-methylenePGE₂, 9-keto Fluprostenol, 5-trans PGE₂, 17-phenyl-omega-trinor PGE₂,PGE₂ serinol amide, PGE₂ methyl ester, 16-phenyl tetranor PGE₂,15(S)-15-methyl PGE₂, 15(R)-15-methyl PGE₂, 8-iso-15-keto PGE₂, 8-isoPGE₂ isopropyl ester, 8-iso-16-cyclohexyl-tetranor PGE₂, 20-hydroxyPGE₂, 20-ethyl PGE₂, 11-deoxy PGEi, nocloprost, sulprostone, butaprost,15-keto PGE₂, or 19 (R) hydroxy PGE₂.
 125. The composition of claim 121,wherein the composition is a culture medium comprising the combination,and further wherein the combination is present in an amount effective tomodulate a population of T cells to (a) reduce expression of one or moreT cell exhaustion markers, or (b) increase mitochondrial sparerespiratory capacity, in comparison to a corresponding population of Tcells that are not modulated with the combination.
 126. The compositionof claim 125, wherein the one or more T cell exhaustion markers compriseone or more of PD-1 and Tim-3.
 127. The composition of claim 125,wherein the combination is present in an amount effective to (a)increase a central memory T cell subpopulation of the population of Tcells, and (b) decrease an effector T cell subpopulation of thepopulation of T cells.
 128. The composition of claim 125, furthercomprising the population of T cells.
 129. The composition of claim 128,wherein the population of T cells comprises (a) increased geneexpression in at least one of CD27, C-C chemokine receptor type 7(CCR7), CD62L, transcription factor 7 (TCF7), lymphoid enhancer-bindingfactor 1 (LEF1), and (b) decreased gene expression in at least one of PRdomain zinc finger protein 1 (BLIMP-1), fructose-bisphosphate aldolase C(ALDOC), gamma enolase (ENO2), PD-1 and Tim-3, in comparison to acorresponding population of T cells that are not modulated with thecombination.
 130. The composition of claim 128, wherein the populationof T cells comprises increased spare respiratory capacity (SRC) incomparison to a corresponding population of T cells that are notmodulated with the composition.
 131. The composition of claim 128,wherein the population of T cells comprises at least one of thefollowing: (a) increased gene expression in at least one of CD27, CCR7,CD62L, TCF7, and LEF1; (b) decreased gene expression in at least one ofBLIMP-1, ALDOC, ENO2, and PGK1; (c) increased central memory T cellsubpopulation; (d) decreased effector T cell subpopulation; and (e)improved capability in tumor clearance and persistence; compared to acorresponding population of T cells that are not modulated with thecomposition.
 132. The composition of claim 128, wherein T cells of thepopulation of T cells comprise at least one genetic modification. 133.The composition of claim 132, wherein the at least one geneticmodification comprises an insertion, a deletion, or a nucleic acidreplacement.
 134. The composition of claim 132, wherein the at least onegenetic modification comprises an exogenous nucleic acid encoding a TCell Receptor (TCR) and/or a Chimeric Antigen Receptor (CAR).
 135. Thecomposition of claim 132, wherein the at least one genetic modificationcomprises insertion or modification of a sequence encoding at least oneof a safety switch protein, a targeting modality, a receptor, asignaling molecule, a transcription factor, a pharmaceutically activeprotein or peptide, a drug target candidate, or a protein promoting oneor more activities; wherein the one or more activities comprise one ormore of engraftment, trafficking, homing, viability, self-renewal,persistence, immune response regulation and modulation, and survival ofthe immune cells.
 136. The composition of claim 132, wherein the atleast one genetic modification comprises deletion or reduced expressionof B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA,RFX5, or RFXAP, or any gene in the chromosome 6p21 region.
 137. Thecomposition of claim 132, wherein the at least one genetic modificationcomprises introduced or increased expression of HLA-E, HLA-G, HACD16,hnCD16, 41BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1,A2AR, Fc receptor, or surface triggering receptors for coupling with bi-or multi-specific or universal engagers.
 138. The composition of claim132, wherein the population of T cells are differentiated in vitro fromstem cells, hematopoietic stem or progenitor cells, or progenitor cells;and wherein the stem cells, hematopoietic stem or progenitor cells, orprogenitor cells comprise the at least one genetic modification. 139.The composition of claim 138, wherein the stem cells comprise inducedpluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).
 140. Thecomposition of claim 138, wherein the progenitor cells are CD34+hemogenic endothelium cells, multipotent progenitor cells, or T cellprogenitor cells.